Dalila Azzout-Marniche

Increasing Protein at the Expense of Carbohydrate in the Diet Down-Regulates Glucose Utilization as Glucose Sparing Effect in Rats

High protein (HP) diet could serve as a good strategy against obesity, provoking the changes in energy metabolic pathways. However, those modifications differ during a dietary adaptation. To better understand the mechanisms involved in effect of high protein diet (HP) on limiting adiposity in rats we studied in parallel the gene expression of enzymes involved in protein and energy metabolism and the profiles of nutrients oxidation. Eighty male Wistar rats were fed a normal protein diet (NP, 14% of protein) for one week, then either maintained on NP diet or assigned to a HP diet (50% of protein) for 1, 3, 6 and 14 days. mRNA levels of genes involved in carbohydrate and lipid metabolism were measured in liver, adipose tissues, kidney and muscles by real time PCR. Energy expenditure (EE) and substrate oxidation were measured by indirect calorimetry. Liver glycogen and plasma glucose and hormones were assayed. In liver, HP feeding 1) decreased mRNA encoding glycolysis enzymes (GK, L-PK) and lipogenesis enzymes(ACC, FAS), 2) increased mRNA encoding gluconeogenesis enzymes (PEPCK), 3) first lowered, then restored mRNA encoding glycogen synthesis enzyme (GS), 4) did not change mRNA encoding β-oxidation enzymes (CPT1, ACOX1, βHAD). Few changes were seen in other organs. In parallel, indirect calorimetry confirmed that following HP feeding, glucose oxidation was reduced and fat oxidation was stable, except during the 1st day of adaptation where lipid oxidation was increased. Finally, this study showed that plasma insulin was lowered and hepatic glucose uptake was decreased. Taken together, these results demonstrate that following HP feeding, CHO utilization was increased above the increase in carbohydrate intake while lipogenesis was decreased thus giving a potential explanation for the fat lowering effect of HP diets.

High dietary protein decreases fat deposition induced by high-fat and high-sucrose diet in rats

High-protein diets are known to reduce adiposity in the context of high carbohydrate and Western diets. However, few studies have investigated the specific high-protein effect on lipogenesis induced by a high-sucrose (HS) diet or fat deposition induced by high-fat feeding. We aimed to determine the effects of high protein intake on the development of fat deposition and partitioning in response to high-fat and/or HS feeding. A total of thirty adult male Wistar rats were assigned to one of the six dietary regimens with low and high protein, sucrose and fat contents for 5 weeks. Body weight (BW) and food intake were measured weekly. Oral glucose tolerance tests and meal tolerance tests were performed after 4th and 5th weeks of the regimen, respectively. At the end of the study, the rats were killed 2 h after ingestion of a calibrated meal. Blood, tissues and organs were collected for analysis of circulating metabolites and hormones, body composition and mRNA expression in the liver and adipose tissues. No changes were observed in cumulative energy intake and BW gain after 5 weeks of dietary treatment. However, high-protein diets reduced by 20 % the adiposity gain induced by HS and high-sucrose high-fat (HS-HF) diets. Gene expression and transcriptomic analysis suggested that high protein intake reduced liver capacity for lipogenesis by reducing mRNA expressions of fatty acid synthase (fasn), acetyl-CoA carboxylase a and b (Acaca and Acacb) and sterol regulatory element binding transcription factor 1c (Srebf-1c). Moreover, ketogenesis, as indicated by plasma β-hydroxybutyrate levels, was higher in HS-HF-fed mice that were also fed high protein levels. Taken together, these results suggest that high-protein diets may reduce adiposity by inhibiting lipogenesis and stimulating ketogenesis in the liver.

Dietary Proteins Contribute Little to Glucose Production Even Under Optimal Gluconeogenic Conditions in Healthy Humans

Dietary proteins are believed to participate significantly in maintaining blood glucose levels, but their contribution to endogenous glucose production (EGP) remains unclear. We investigated this question using multiple stable isotopes. After overnight fasting, eight healthy volunteers received an intravenous infusion of [6,6-2H2]-glucose. Two hours later, they ingested four eggs containing 23 g of intrinsically, uniformly, and doubly [15N]-[13C]–labeled proteins. Gas exchanges, expired CO2, blood, and urine were collected over the 8 h following egg ingestion. The cumulative amount of dietary amino acids (AAs) deaminated over this 8-h period was 18.1 ± 3.5%, 17.5% of them being oxidized. The EGP remained stable for 6 h but fell thereafter, concomitantly with blood glucose levels. During the 8 h after egg ingestion, 50.4 ± 7.7 g of glucose was produced, but only 3.9 ± 0.7 g originated from dietary AA. Our results show that the total postprandial contribution of dietary AA to EGP was small in humans habituated to a diet medium-rich in proteins, even after an overnight fast and in the absence of carbohydrates from the meal. These findings question the respective roles of dietary proteins and endogenous sources in generating significant amounts of glucose in order to maintain blood glucose levels in healthy subjects.

[13C] GC–C-IRMS analysis of methylboronic acid derivatives of glucose from liver glycogen after the ingestion of [13C] labeled tracers in rats

We developed a complete method to measure low [(13)C] enrichments in glycogen. Fourteen rats were fed a control diet. Six of them also ingested either [U-(13)C] glucose (n=2) or a mixture of 20 [U-(13)C] amino acids (n=4). Hepatic glycogen was extracted, digested to glucose and purified on anion-cation exchange resins. After the optimization of methylboronic acid derivatization using GC-MS, [(13)C] enrichment of extracted glucose was measured by GC-C-IRMS. The accuracy was addressed by measuring the enrichment excess of a calibration curve, which observed values were in good agreement with the expected values (R=0.9979). Corrected delta values were -15.6+/-1.6 delta(13)C (per thousand) for control rats (n=8) and increased to -5 to 8 delta(13)C (per thousand) per thousand and 12-14 delta(13)C (per thousand) per thousand after the ingestion of [U-(13)C] amino acids or [U-(13)C] glucose as oral tracers, respectively. The method enabled the determination of dietary substrate transfer into glycogen. The sequestration of dietary glucose in liver glycogen 4 h after the meal was 35% of the ingested dose whereas the transfer of carbon skeletons from amino acids was only 0.25 to 1%.

Adaptation to a high-protein diet progressively increases the postprandial accumulation of carbon skeletons from dietary amino acids in rats

We aimed to determine whether oxidative pathways adapt to the overproduction of carbon skeletons resulting from the progressive activation of amino acid (AA) deamination and ureagenesis under a high-protein (HP) diet. Ninety-four male Wistar rats, of which 54 were implanted with a permanent jugular catheter, were fed a normal protein diet for 1 wk and were then switched to an HP diet for 1, 3, 6, or 14 days. On the experimental day, they were given their meal containing a mixture of 20 U-[15N]-[13C] AA, whose metabolic fate was followed for 4 h. Gastric emptying tended to be slower during the first 3 days of adaptation. 15N excretion in urine increased progressively during the first 6 days, reaching 29% of ingested protein. 13CO2 excretion was maximal, as early as the first day, and represented only 16% of the ingested proteins. Consequently, the amount of carbon skeletons remaining in the metabolic pools 4 h after the meal ingestion progressively increased to 42% of the deaminated dietary AA after 6 days of HP diet. In contrast, 13C enrichment of plasma glucose tended to increase from 1 to 14 days of the HP diet. We conclude that there is no oxidative adaptation in the early postprandial period to an excess of carbon skeletons resulting from AA deamination in HP diets. This leads to an increase in the postprandial accumulation of carbon skeletons throughout the adaptation to an HP diet, which can contribute to the sustainable satiating effect of this diet.

Low-protein diet-induced hyperphagia and adiposity are modulated through interactions involving thermoregulation, motor activity, and protein quality in mice

Low protein (LP)-containing diets can induce overeating in rodents and possibly in humans in an effort to meet protein requirement, but the effects on energy expenditure (EE) are unclear. The present study evaluated the changes induced by reducing dietary protein from 20% to 6%-using either soy protein or casein-on energy intake, body composition, and EE in mice housed at 22°C or at 30°C (thermal neutrality). LP feeding increased energy intake and adiposity, more in soy-fed than in casein-fed mice, but also increased EE, thus limiting fat accumulation. The increase in EE was due mainly to an increase in spontaneous motor activity related to EE and not to thermoregulation. However, the high cost of thermoregulation at 22°C and the subsequent heat exchanges between nonshivering thermogenesis, motor activity, and feeding induced large differences in adaptation between mice housed at 22°C and at 30°C.

Metabolic effects of intermittent access to caloric or non-caloric sweetened solutions in mice fed a high-caloric diet

Human consumption of obesogenic diets and soft drinks, sweetened with different molecules, is increasing worldwide, and increases the risk of metabolic diseases. We hypothesized that the chronic consumption of caloric (sucrose, high-fructose corn syrup (HFCS), maltodextrin) and non-caloric (sucralose) solutions under 2-hour intermittent access, alongside the consumption of a high-fat high-sucrose diet, would result in differential obesity-associated metabolic abnormalities in mice. Male C57BL/6 mice had ad libitum access to an HFHS diet and to water (water control group). In addition, some mice had access, 2h/day, 5days/week (randomly chosen) for 12weeks, to different solutions: i) a sucrose solution (2.1kJ/ml), ii) an HFCS solution (2.1kJ/ml), iii) a maltodextrin solution (2.1kJ/ml) and a sucralose solution (60mM) (n=15/group). Despite no changes in total caloric intake, 2h-intermittent access to the sucrose, HFCS or maltodextrin solutions led to increased body weight and accumulation of lipids in the liver when compared to the group consuming water only. The HFCS and sucrose solutions induced a higher fat mass in various fat depots, glucose intolerance, increased glucose oxidation at the expense of lipid oxidation, and a lower hypothalamic expression of NPY in the fasted state. HFCS also reduced proopiomelanocortin expression in the hypothalamus. 2h-intermittent access to sucralose did not result in significant changes in body composition, but caused a stronger expression of CART in the hypothalamus. Finally, sucrose intake showed a trend to increase the expression of various receptors in the nucleus accumbens, linked to dopamine, opioid and endocannabinoid signaling. In conclusion, 2h-intermittent access to caloric solutions (especially those sweetened with sucrose and HFCS), but not sucralose, resulted in adverse metabolic consequences in high-fat high-sucrose-fed mice.