A fish oil diet does not reverse insulin resistance despite decreased adipose tissue TNF-alpha protein concentration in ApoE-3*Leiden mice

Dietary interventions with fish oil have been found to protect against the development of high-fat diet-induced insulin resistance and to decrease the expression of tumor necrosis factor (TNF)-alpha. However, the effect of fish oil administration on preexisting insulin resistance is subject to debate. In the present study, we examined the mechanism by which fish oil affects preexisting insulin resistance. High fat diet-induced insulin-resistant ApoE*3-Leiden transgenic mice were treated for 10 wk as follows: 1) high fat diet (control group), 2) high fat diet with 3 g/100 g fish oil and 3) high fat diet but food intake restricted to 75% of the ad libitum food intake. We measured plasma glucose, insulin, free fatty acids (FFA) and triglyceride (TG) levels throughout the study. After the 10-wk dietary intervention period we performed hyperinsulinemic euglycemic analyses and measured insulin sensitivity and FFA turnover. Furthermore, we then determined the VLDL-TG production rate and TNF-alpha protein expression in white adipose tissue (WAT). Compared with control mice, the insulin sensitivity of mice treated with fish oil was not affected, whereas it was improved (P < 0.05) for energy-restricted mice. FFA turnover was unaffected in both fish oil-treated and energy-restricted mice. Compared with controls, hepatic VLDL-TG production was lower (P < 0.05) with fish oil feeding but greater with energy restriction (P < 0.05). Interestingly, the level of TNF-alpha protein in WAT was lower (P < 0.05) in both groups. We conclude that partial replacement of saturated fat by fish oil does not improve preexisting high fat diet-induced insulin resistance, although it lowers TNF-alpha protein levels in WAT.     

Varying the protein source in mixed meal modifies glucose, insulin and glucagon kinetics in healthy men, has weak effects on subjective satiety and fails to affect food intake

OBJECTIVE: To evaluate the influence of three dietary protein types (casein, gelatin, soy protein) on satiety and food intake, at two levels of loading (total energy of test meals: 3.6 or 1.8 MJ). DESIGN: The study employed a repeated measures design. Test meals were controlled for energy, macronutrients, fiber and palatability, and contained about 23% energy as protein (of which about 65% was experimentally manipulated). Postprandial subjective satiety and hunger, plasma glucose, insulin and glucagon were assessed for 8 h, and energy and macronutrient intakes were monitored for 24 h. SUBJECTS: Nine healthy normal-weight men. RESULTS: No effect of the type of protein on 24 h energy and macronutrient intakes was observed despite a significant effect of protein source on the kinetics of peripheral metabolic responses (but only after 3.6 MJ lunches), and inconsistent effects on subjective hunger and satiety responses A casein-enriched lunch delayed glucose and insulin responses for 1.5 h, compared with soy protein, probably due to a lag in gastric emptying. CONCLUSION: Varying the protein source in a mixed meal modifies glucose, insulin and glucagon kinetics in healthy men, but these variations in satiety-implicated factors have inconsistent effects on subjective satiety and fail to affect food intake. SPONSORSHIP: Eridania Béghin-Say, Vilvoorde, Belgium and Association Nationale de la Recherche Technique, France (Convention CIFRE no 537/94).     

Effects of high-fat diet and physical activity on pyruvate dehydrogenase kinase-4 in mouse skeletal muscle

Background

The expression of PDK4 is elevated by diabetes, fasting and other conditions associated with the switch from the utilization of glucose to fatty acids as an energy source. It is previously shown that peroxisome proliferator-activated receptor ?? coactivator 1?? (PGC-1??), a master regulator of energy metabolism, coactivates in cell lines pyruvate dehydrogenase kinase-4 (PDK4) gene expression via the estrogen-related receptor ?? (ERR??). We investigated the effects of long-term high-fat diet and physical activity on the expression of PDK4, PGC-1?? and ERR?? and the amount and function of mitochondria in skeletal muscle.

Methods

Insulin resistance was induced by a high-fat (HF) diet for 19?weeks in C57BL/6?J mice, which were either sedentary or with access to running wheels. The skeletal muscle expression levels of PDK4, PGC-1?? and ERR?? were measured and the quality and quantity of mitochondrial function was assessed.

Results

The HF mice were more insulin-resistant than the low-fat (LF) -fed mice. Upregulation of PDK4 and ERR?? mRNA and protein levels were seen after the HF diet, and when combined with running even more profound effects on the mRNA expression levels were observed. Chronic HF feeding and voluntary running did not have significant effects on PGC-1?? mRNA or protein levels. No remarkable difference was found in the amount or function of mitochondria.

Conclusions

Our results support the view that insulin resistance is not mediated by the decreased qualitative or quantitative properties of mitochondria. Instead, the role of PDK4 should be contemplated as a possible contributor to high-fat diet-induced insulin resistance.     

Response of hormones modulating plasma cholesterol to dietary casein or soy protein in minipigs

To elucidate the mechanism mediating the effect of dietary casein or soy protein on serum cholesterol concentrations we followed the endocrine response to the intake of these dietary proteins. The hormones analyzed were those known to modulate serum cholesterol concentration. A 7-wk crossover nutrition study was performed with six adult G?ttingen minipigs consuming semisynthetic diets based on either 20 wt% casein or soy isolate. At d 42 and 49, concentrations of six hormones were determined in 22 blood samples taken over the whole day. There were no significant differences in insulin, glucagon, the insulin/glucagon ratio, hydrocortisone or triiodothyronine among dietary groups. In the late postprandial phase (5 h after the meal and later) there were significantly higher growth hormone concentrations in soy-fed animals. At all times of the day, total and free thyroxine concentrations were higher after soy feeding than after casein feeding. On average, total and free thyroxine concentrations were 34 and 26% higher with soy protein feeding than with casein feeding. Our data agree with other reports of protein-dependent changes of thyroid hormones and may explain why different dietary proteins have different effects on serum cholesterol levels in sensitive species.     

Specific decrease in liver insulin-like growth factor-I and brain insulin-like growth factor-II gene expression in energy-restricted rats.

Four-week-old male rats were maintained for 10 d on a series of diets containing a constant high level of dietary protein and total energy at 100, 70, 60 or 50% of the ad libitum intake rate. Under these conditions, growth rate varied as a function of dietary energy. Serum insulin-like growth factor (IGF)-I was decreased in the energy-restricted animals. Total hepatic IGF-I mRNA was decreased by approximately the same factor as circulating IGF-I protein. In contrast to previous results obtained with protein-restricted animals, serum albumin mRNA was not decreased in the energy-restricted animals. Brain IGF-II mRNA was slightly decreased in animals fed the 70 and 60% energy diets and was decreased by 50% in animals fed the 50% energy diet. Insulin-like growth factor binding protein-2 (IGFBP-2) gene expression was increased in the liver but not in the brain of the energy-restricted animals, indicating that dietary energy regulates IGFBP-2 gene expression differently in liver and brain. The results demonstrate specific changes in liver IGF-I and IGFBP-2 gene expression and brain IGF-II gene expression in animals that are growth-retarded because of a restriction of dietary energy.     

大豆蛋白对高脂饲料喂养大鼠固醇调节元件结合蛋白基因表达的影响

目的探讨大豆蛋白在正常饮食和高脂饮食状态下对大鼠脂代谢相关固醇调节元件结合蛋白(SREBP)基因表达水平的影响。方法 48只清洁级SD大鼠,按体重随机分为4组,分别喂饲含酪蛋白和大豆蛋白的正常饲料和高脂饲料28d后,脱颈椎处死动物,检测激素指标及基因表达水平。结果大豆蛋白组大鼠血清胰岛素水平和胰岛素/胰高血糖素比值显著低于酪蛋白组(P<0.05);大豆蛋白高脂组大鼠血清胰岛素水平显著低于酪蛋白高脂组(P<0.05);大豆蛋白组和大豆蛋白高脂组大鼠肝脏SREBP-1,2基因表达均分别显著低于酪蛋白组和酪蛋白高脂组(P<0.05);大豆蛋白组和大豆蛋白高脂组大鼠肝脏SREBP-1蛋白表达明显低于酪蛋白组和酪蛋白高脂组(P<0.05)。结论大豆蛋白可能通过影响胰岛素进而影响SREBP-1基因表达来调节血脂水平。     

Gene expressions of leptin, insulin receptors and lipogenic enzymes are coordinately regulated by insulin and dietary fat in rats

Regulation of the gene expressions of leptin, insulin receptors and lipogenic enzymes was investigated after refeeding a fat-free diet or a 10 g/100 g corn oil diet to food-deprived rats. Plasma glucose and insulin concentrations began to increase 30 min after the feeding and further increased up until 8 h. In these rats, the expression of leptin mRNA in adipose tissue began to increase significantly only 30 min after feeding, and reached a maximum at 8-16 h. However, plasma leptin levels did not increase until 4 h after refeeding, then markedly increased and reached the maximal level after 8 h. The expression of leptin mRNA and plasma leptin concentrations generally were greater in rats fed the corn oil diet compared to those fed the fat-free diet. Insulin receptor mRNA concentrations in the liver and adipose tissue began to decrease 30 min after the refeeding, in contrast to the plasma insulin increase, and continued to decrease until 8 h. The expression of acetyl-CoA carboxylase and fatty acid synthase mRNA began to increase 4-8 h after feeding and reached maximal levels at 16-24 h. Leptin treatment suppressed the expression of lipogenic enzyme mRNA in rats fed the fat-free diet but not in corn oil-fed rats, in which the expression was suppressed by polyunsaturated fatty acids and leptin expression was higher. Thus, we suggest that the glucose and insulin-dependent expressions of leptin, insulin receptors and lipogenic enzymes are coordinately and/or mutually regulated by dietary manipulation.     

Effects of energy restriction on mouse mammary tumor virus mRNA levels in mammary glands and uterus and on uterine endometrial hyperplasia and pituitary histology in C3H/SHN F1 mice

We investigated the effects of energy restriction on the pituitary-ovarian axis and on a hormone responsive gene, the mouse mammary tumor virus (MMTV). Female C3H/SHN F1-hybrid mice, known to display a high incidence of mammary tumors, ate an energy-restricted diet (48 kcal/wk) or a control diet (95 kcal/wk) beginning at the time of weaning. By 67 wk of age, 12 of 32 mice in the control group, but none of the 33 mice in the energy-restricted group, had developed mammary tumors. Six tumor-free mice from each group were studied in detail at 67 wk of age. All six tumor-free control mice, but none of the six energy-restricted mice, showed uterine endometrial hyperplasia at autopsy. Mice subjected to energy restriction did not display an estrous cycle. The average levels of MMTV mRNA in mammary glands and uteri were strongly reduced by energy restriction. MMTV mRNA levels in mammary glands from control mice were two orders of magnitude lower than those in mammary tumors. Energy restriction lowered the percentage of pituitary mammatropes and suppressed proliferation of mammatropes with advancing age. Energy restriction thus appeared to inhibit endometrial hyperplasia and to decrease MMTV production at the mRNA level in the mammary glands and in the uterus. These effects may be a consequence of hormonal changes originating at the pituitary-ovarian axis.     

Adipocyte gene expression is altered in formerly obese mice and as a function of diet composition

In the development of obesity, the source of excess energy may influence appetite and metabolism. To determine the effects of differences in diet composition in obesity, mice were fed either a high-carbohydrate diet (HC; 10% fat energy) or a high-fat energy-restricted diet (HFR; 60% fat energy) over 18 wk in weight-matched groups of mice. To identify obesity-associated genes with persistently altered expression following weight reduction, mice were fed either a standard low-fat diet (LF; 10% fat energy), an unrestricted high-fat diet (HF; 60% fat energy), or a HF diet followed by weight reduction (WR). Mice fed a HF diet had significantly greater gonadal fat mass and higher whole blood glucose concentrations than mice fed an HC diet. Of the mice fed a high-fat diet, total body weight and serum insulin concentrations were greater in HF than in HFR. Microarray analysis revealed that HF vs. HC feeding resulted in global differences in adipocyte gene expression patterns. Although we identified genes whose expression was altered in both moderately and severely obese mice, there were also a large number of genes with altered expression only in severe obesity. Formerly obese, WR mice did not differ significantly from lean controls in total body weight or physiological measures. However, microarray analysis revealed distinctly different patterns of adipocyte gene expression. Furthermore, there were 398 genes with altered expression in HF mice that persisted in WR mice. Genes with persistently altered expression following obesity may play a role in rebound weight gain following weight reduction.     

The effect of protein ingestion on the metabolic response to oral glucose in normal individuals

Eight normal subjects were given 50 g protein, 50 g glucose, or 50 g protein + 50 g glucose. Plasma glucose, insulin, C-peptide, glucagon, alpha-amino nitrogen (AAN), and nonesterified fatty acid (NEFA) responses were then determined over 4 h. Protein stimulated only a modest insulin rise and the area above fasting baseline was only 28% of that after glucose. The sum of the serum insulin area following protein ingestion and that following glucose ingestion was 100.4% of the combination meal. C-peptide changes confirmed the insulin response. The addition of glucose to the protein meal resulted in a 60 min delay in glucagon and AAN rise compared to the protein meal alone. Subsequently AAN and glucagon increased to levels greater than or equal to those observed after protein ingestion alone. In summary, protein is a much less potent secretagogue for insulin than is glucose in normal individuals, and the effect on insulin secretion is not synergistic. Addition of glucose to a protein meal results in a delayed rise in AAN and glucagon concentrations in normal subjects.     

禁食和再喂养期间大鼠小肠胰岛素和胰岛素样生长因子-1受体的比较

胰岛素样生长因子-1（IGF-1）和胰岛素可能是肠道生长的重要调节因子。为了研究肠细胞萎缩和再生期间小肠IGF－1受体（IGF-IR）和胰岛素受体（IR），我们比较了禁食72小时和肠内再喂养24～72小时大鼠空肠IGF-IR和IR表达的指标。禁食引起肠萎缩，血浆胰岛素和IGF－1浓度降低以及空肠IGF-1信使RNA（mRNA）水平的明显降低，再喂养可逆转这些改变。禁食明显增加胰岛素与空肠特异性地结合，IR含量（达对照组的230％）和9．6kb和7．4kbIRmRNA转录本水平（分别达对照组的202％和218％）。再喂养时，这些IR指标迅速降到对照组水平。禁食时IGP-IR（用Scatchard分析）和IGF－1－RmRNA无明显的改变。再喂养后的前24小时间11-kbIGF－IRmRNA转录本明显增加（达对照组水平166％），IGF-IR数量增加3倍。我们的结论是：大鼠空肠的IR和IGF－IR受到不同营养物利用状态的调节。再喂养时空肠IGF－1和IGF－IR表达的向上调节表明，IGF作用途径在对肠内营养物产生肠道营养反应的过程中起作用。     

Gender Differences in Response to Prolonged Every-Other-Day Feeding on the Proliferation and Apoptosis of Hepatocytes in Mice

Intermittent fasting decreases glucose and insulin levels and increases insulin sensitivity and lifespan. Decreased food intake influences the liver. Previous studies have shown gender differences in response to various types of caloric restriction, including every-other-day (EOD) feeding, in humans and rodents. Our goal was to show the influence of prolonged EOD feeding on the morphology, proliferation and apoptosis of livers from male and female mice. After nine months of an EOD diet, the livers from male and female mice were collected. We examined their morphology on histological slides using the Hematoxilin and Eosine (H_E) method and Hoechst staining of cell nuclei to evaluate the nuclear area of hepatocytes. We also evaluated the expression of mRNA for proto-oncogens, pro-survival proteins and apoptotic markers using Real Time Polimerase Chain Reaction (PCR). We noted increased lipid content in the livers of EOD fed female mice. EOD feeding lead to a decrease of proliferation and apoptosis in the livers of female and male mice, which suggest that tissue maintenance occurred during EOD feeding. Our experiment revealed sex-specific expression of mRNA for proto-oncogenes and pro-survival and pro-apoptotic genes in mice as well as sex-specific responses to the EOD treatment.     

Dietary components and plasma insulin responses to fasting and refeeding in genetically obese hyperglycaemic (ob/ob) mice

To investigate the role of dietary components in the hyperinsulinaemia of the obese hyperglycaemic (ob/ob) syndrome, plasma insulin responses to fasting and refeeding were examined in Aston ob/ob mice supplied with standard diet, non-digestible-carbohydrate test food, and isoenergetic test foods from which either carbohydrate, protein or fat was omitted. During fasting, plasma insulin concentrations fell more rapidly and to a greater extent than plasma glucose. Refeeding the standard diet raised insulin concentrations above normal, associated with a 25% compensatory increase in food intake over 24 h. Test foods supplied to previously fed or fasted mice produced glucose responses consistent with the available carbohydrate content. Carbohydrate-free food (protein and fat) provided a small insulinotropic stimulus; the effect of protein-free food (carbohydrate and fat) was greater; and the combination of carbohydrate with protein (fat-free food) evoked a marked insulin response. In contrast, insulin concentrations declined in mice given the non-digestible-carbohydrate food. Consumption of the standard diet was increased after 24 h feeding non-digestible-carbohydrate food, but was unaffected by a 30 h fast initiated 54 h previously. These results demonstrate that hyperinsulinaemia in ob/ob mice is not merely triggered by the ingestion of bulk, but depends on the type of nutrient ingested. Dietary carbohydrate appears to be the major stimulus to the hyperinsulinaemia, with an important augmentation in the presence of protein. Since direct glucose stimulation of insulin release is defective in ob/ob mice, the hyperinsulinaemia must be mediated by increased activity of the enteroinsular axis.     

The small intestine proteome is changed in preterm pigs developing necrotizing enterocolitis in response to formula feeding

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in newborn premature infants. Clinical studies show increased incidence of NEC in premature infants with enteral formula feeding; however, pathogenesis remains unclear. To identify the NEC-related proteins for molecular mechanisms, we applied proteomics analysis to characterize changes in the protein expression profile of newborn premature piglet intestines with NEC developed after enteral formula feeding for 24 h. Changes in protein expression were identified using 2-dimensional gel electrophoresis and peptide mass fingerprinting with MS as well as western blotting analysis. Nineteen differentially expressed proteins were identified and these have roles in oxidative stress, chaperone, signal transduction, protein folding and degradation, oxygen transport, signal transduction, and energy metabolism. Proteins with increased levels include manganese-containing superoxide dismutase and hemoglobin subunit and proteins with decreased expression include sorbitol dehydrogenase, mitochondrial aldehyde dehydrogenase 2, glucose-regulated protein 75, CRY protein, snail homolog 3, thyroid hormone-binding protein precursor, and DJ1 (Parkinson's disease 7) etc. The data provided novel mechanistic insights into the pathogenesis of NEC and the insults of a formulated diet to the premature gut.     

Dietary protein concentration regulates the mRNA expression of chicken hepatic malic enzyme

Chicken hepatic malic enzyme activity varies with dietary protein content. The mechanisms responsible for this alteration in activity are unclear. In a series of four experiments, broiler chicks were allowed free access for 1.5, 3, 6 or 24 h to a low (13 g/100 g diet), basal (22 g/100 g diet) or high (40 g/100 g diet) protein diet. The diets were isocaloric and had equal concentrations of dietary fat. Hepatic malic enzyme mRNA expression and enzyme activity as well as total liver lipid concentration were examined for each experimental duration. There were no differences in the expression of the mRNA for malic enzyme at 1.5 h, but at 3, 6 and 24 h, malic enzyme mRNA expression was significantly (P < 0.05) reduced in chicks fed the high protein diet and significantly enhanced in chicks fed the low protein diet compared with chicks fed the basal diet. Hepatic malic enzyme activities and total lipid concentration were not different among the chicks fed the different diets at 1.5 and 3 h. At 6 and 24 h, malic enzyme activity and total liver lipid concentration were both significantly greater in birds fed the low protein diet compared with levels in the birds fed the other two diets. In birds fed the high protein diet, malic enzyme activity and total liver lipid concentration were significantly reduced at 24 h compared with birds fed the basal diet. In a final experiment, the observed differences in malic enzyme mRNA expression at 6 h were confirmed when chicks were given access to isocaloric diets with the same protein levels as the initial 4 experiments, but with the dietary concentration of carbohydrate held constant. The results suggest that previously observed alterations in the activity of malic enzyme, which were correlated with dietary protein intake, are due to rapid changes in the mRNA expression of this enzyme.     

Conjugated linoleic acid deteriorates insulin resistance in obese/diabetic mice in association with decreased production of adiponectin and leptin

Dietary supplementation of conjugated linoleic acids (CLA) is known to have some beneficial effects such as anti-carcinogenic and anti-obesity effects in several animal species, while it also induces insulin resistance and fatty liver, especially in mice. To explore the possible factors responsible for the CLA-induced insulin resistance, we examined the plasma and mRNA expression levels of several adipocytokines, which are likely involved in the regulation of insulin sensitivity, in normal C5 7BL, mildly obese/diabetic KK and morbidly obese/diabetic KKAy mice. Feeding a diet supplemented with 0.5%, CLA oil consisting of 30.5/% c9, t11-CLA and 28.9% t10, c12-CLA for 4 wk resulted in a decrease in white adipose tissue (WAT), an increase in liver weight with excess accumulation of triglyceride, and insulin resistance associated with hyperglycemia and hyperinsulinemia. The plasma and WAT mRNA levels of leptin were higher in KK and KKAy mice than C57BI. mice, whereas those of adiponectin were higher in C5 7BL mice. CLA-feeding decreased the levels of leptin, adiponectin and resistin, especially in KK and KKAy mice. In contrast, tumor necrosis factor-alpha (TNFalpha) mRNA levels were higher in KK and KKAy mice than C57BL mice, and were increased by CLA feeding. The present results thus indicate that CLA feeding promotes insulin resistance in obese/diabetic mice by at least inverse regulation of leptin and adiponectin, and TNFalpha, adipocytokines known to either ameliorate or deteriorate insulin sensitivity, respectively.