Consumption of a Carbonated Beverage with High-Intensity Sweeteners Has No Effect on Insulin Sensitivity and Secretion in Nondiabetic Adults.

Background: The effects of the regular intake of beverages containing high-intensity sweeteners on insulin sensitivity in healthy individuals remain controversial. Objective: This trial compared the effects of the consumption of a carbonated beverage containing aspartame and acesulfame K (high-intensity sweeteners beverage-HISB) with those of an unsweetened, no-calorie carbonated beverage (UB) on insulin sensitivity and secretion in nondiabetic adults. Methods: SEDULC was a randomized, double-blind, crossover study. Nondiabetic adults [mean age 31 y, 44% men, body mass index (BMI; kg/m²) 19-29] who did not consume high-intensity sweeteners were randomized 1:1 to drink 1 of the 2 carbonated beverages, 2 cans (330 mL each)/d, for 12 wk. After a 4-wk washout period, participants were switched to the opposite beverage for 12 wk. The primary outcome tested was the change in insulin sensitivity as assessed by the Matsuda Insulin Sensitivity Index (MISI) after an oral glucose load. Secondary outcomes were indexes of insulin secretion. Results: Sixty individuals were enrolled and 50 completed the study (28 nonoverweight and 22 overweight participants). The change in MISI from baseline did not significantly differ between beverages and noninferiority was demonstrated (difference = -0.23; 95% CI: -1.31, 0.85; P < 0.0001). The change in insulinogenic (means ± SEMs: 0.23 ± 0.14 for HISB compared with 0.08 ± 0.1 for UB) and disposition indexes (2.70 ± 0.99 for HISB compared with 1.62 ± 0.90 for UB) did not differ, and no differences in insulin secretion estimates were confirmed by the Stumvoll indexes. Consuming the high-intensity sweeteners did not affect body weight, self-reported dietary consumption, or self-reported physical activity. Conclusions: These findings suggest that the daily consumption of 2 cans of a beverage containing aspartame and acesulfame K over 12 wk has no significant effect on insulin sensitivity and secretion in nondiabetic adults. This trial was registered at clinicaltrials.gov as NCT02031497.

Vitamin D3 Prevents Calcium-Induced Progression of Early-Stage Prostate Tumors by Counteracting TRPC6 and Calcium Sensing Receptor Upregulation.

Active surveillance has emerged as an alternative to immediate treatment for men with low-risk prostate cancer. Accordingly, identification of environmental factors that facilitate progression to more aggressive stages is critical for disease prevention. Although calcium-enriched diets have been speculated to increase prostate cancer risk, their impact on early-stage tumors remains unexplored. In this study, we addressed this issue with a large interventional animal study. Mouse models of fully penetrant and slowly evolving prostate tumorigenesis showed that a high calcium diet dramatically accelerated the progression of prostate intraepithelial neoplasia, by promoting cell proliferation, micro-invasion, tissue inflammation, and expression of acknowledged prostate cancer markers. Strikingly, dietary vitamin D prevented these calcium-triggered tumorigenic effects. Expression profiling and in vitro mechanistic studies showed that stimulation of PC-3 cells with extracellular Ca2+ resulted in an increase in cell proliferation rate, store-operated calcium entry (SOCE) amplitude, cationic channel TRPC6, and calcium sensing receptor (CaSR) expression. Notably, administration of the active vitamin D metabolite calcitriol reversed all these effects. Silencing CaSR or TRPC6 expression in calcium-stimulated PC3 cells decreased cell proliferation and SOCE. Overall, our results demonstrate the protective effects of vitamin D supplementation in blocking the progression of early-stage prostate lesions induced by a calcium-rich diet. Cancer Res; 77(2); 355-65. ©2016 AACR.

Effect of Increased Water Intake on Urinary DNA Adduct Levels and Mutagenicity in Smokers: A Randomized Study.

UNLABELLED: The association between fluid intake and bladder cancer risk remains controversial. Very little is known about to which extent the amount of water intake influences the action of excreting toxics upon the urinary system. This proof of concept trial investigates the effect of water intake on mutagenesis in smokers, a high risk population for bladder cancer. METHODS: Monocentric randomized controlled trial. Inclusion Criteria. Male subjects aged 2045-45 y/o, smokers, and small drinkers (24-hour urinary volume <1 L and osmolality >700 mOsmol/kg). OUTCOMES: 4-ABP DNA adducts formation in exfoliated bladder cells in 24-hour urine collection and urinary mutagenicity in 24-hour urine. TEST GROUP: Subjects consumed 1.5 L daily of the study product (EVIAN) on top of their usual water intake for 50 days. CONTROL GROUP: Subjects continued their usual lifestyle habits. RESULTS: 65 subjects were randomized. Mean age was 30 y/o and mean cigarettes per day were 20. A slight decrease in adducts formation was observed between baseline and last visit but no statistically significant difference was demonstrated between the groups. Urinary mutagenicity significantly decreased. The study shows that increasing water intake decreases urinary mutagenicity. It is not confirmed by urinary adducts formation. Further research would be necessary.

Hydration Deficit in 9- to 11-Year-Old Egyptian Children.

Background. Children who drink too little to meet their daily water requirements are likely to become dehydrated, and even mild dehydration can negatively affect health. This is even more important in Middle-Eastern countries where high temperatures increase the risk of dehydration. We assessed morning hydration status in a sample of 519 Egyptian schoolchildren (9-11 years old). Methods. Children completed a questionnaire on breakfast intakes and collected a urine sample after breakfast. Breakfast food and fluid nutritional composition was analyzed and urine osmolality was measured using osmometry. Results. The mean urine osmolality of children was 814 mOsmol/kg: >800 mOsmol/kg (57%) and >1000 mOsmol/kg (24.7%). Furthermore, the results showed that a total water intake of less than 400 mL was associated with a significant higher risk of dehydration. Surprisingly, 63% of the children skipped breakfast. Conclusions. The results showed that a majority of Egyptian schoolchildren arrive at school with a hydration deficit. These results highlight the fact that there is a need to educate schoolchildren about the importance of having a breakfast and adequate hydration.

Assessment of hydration status in a large population.

Both acute and chronic dehydration can have important implications for human behaviour and health. Young children, non-autonomous individuals and the elderly are at a greater risk of dehydration. Mild hypertonic dehydration could be related to less efficient cognitive and physical performance and has been reported to be associated with frequently occurring pathological conditions, especially nephrolithiasis. The assessment of hydration status in a large sample appears to be of interest for conducting epidemiological and large clinical studies aimed at improving preventive and curative care. Especially in large-population studies, methods that are used have to be accurate, cheap, quick and require no technical expertise. Body weight change is widely used to determine acute hydration changes, but seems to be insufficiently accurate in longitudinal studies. Bioimpedance analysis methods enable the assessment of total body water content, but their use is still under debate. Because plasma osmolality directly reflects intracellular osmolality, it constitutes a good marker to assess acute hydration changes, but not chronic hydration status because it changes constantly. Moreover, venepuncture is considered to be invasive and is not suitable for a large-sample study, especially in children. Urinary markers appear to be good alternatives for assessing hydration status in large populations. Collection of urine samples is non-invasive and cheap. High technical expertise is not required to perform urinary marker measurements and these measurements can be carried out quickly. Thus, methods based on urinary markers are very well suited for field studies. Urine colour is probably the least sensitive marker despite its high specificity. Urine osmolality and especially urine specific gravity could be easily used for determining hydration status in large-sample studies.

The vesicular glutamate transporter VGLUT3 contributes to protection against neonatal hypoxic stress.

Neonates respond to hypoxia initially by increasing ventilation, and then by markedly decreasing both ventilation (hypoxic ventilatory decline) and oxygen consumption (hypoxic hypometabolism). This latter process, which vanishes with age, reflects a tight coupling between ventilatory and thermogenic responses to hypoxia. The neurological substrate of hypoxic hypometabolism is unclear, but it is known to be centrally mediated, with a strong involvement of the 5-hydroxytryptamine (5-HT, serotonin) system. To clarify this issue, we investigated the possible role of VGLUT3, the third subtype of vesicular glutamate transporter. VGLUT3 contributes to glutamate signalling by 5-HT neurons, facilitates 5-HT transmission and is expressed in strategic regions for respiratory and thermogenic control. We therefore assumed that VGLUT3 might significantly contribute to the response to hypoxia. To test this possibility, we analysed this response in newborn mice lacking VGLUT3 using anatomical, biochemical, electrophysiological and integrative physiology approaches. We found that the lack of VGLUT3 did not affect the histological organization of brainstem respiratory networks or respiratory activity under basal conditions. However, it impaired respiratory responses to 5-HT and anoxia, showing a marked alteration of central respiratory control. These impairments were associated with altered 5-HT turnover at the brainstem level. Furthermore, under cold conditions, the lack of VGLUT3 disrupted the metabolic rate, body temperature, baseline breathing and the ventilatory response to hypoxia. We conclude that VGLUT3 expression is dispensable under basal conditions but is required for optimal response to hypoxic stress in neonates.

French children start their school day with a hydration deficit.

BACKGROUND AND AIMS: Fluid requirements of children vary as a function of gender and age. To our knowledge, there is very little literature on the hydration status of French children. We assessed the morning hydration status in a large sample of 529 French schoolchildren aged 9-11 years. METHODS: Recruited children completed a questionnaire on fluid and food intake at breakfast and collected a urine sample the very same day after breakfast. Breakfast food and fluid nutritional composition was analyzed and urine osmolality was measured using a cryoscopic osmometer. RESULTS: More than a third of the children had a urine osmolality between 801 and 1,000 mosm/kg while 22.7% had a urine osmolality over 1,000 mosm/kg. This was more frequent in boys than in girls (p < 0.001). A majority of children (73.5%) drank less than 400 ml at breakfast. Total water intake at breakfast was significantly and inversely correlated with high osmolality values. CONCLUSIONS: Almost two thirds of the children in this large cohort had evidence of a hydration deficit when they went to school in the morning, despite breakfast intake. Children's fluid intake at breakfast does not suffice to maintain an adequate hydration status for the whole morning.

VGLUT3 (vesicular glutamate transporter type 3) contribution to the regulation of serotonergic transmission and anxiety.

Three different subtypes of H(+)-dependent carriers (named VGLUT1-3) concentrate glutamate into synaptic vesicles before its exocytotic release. Neurons using other neurotransmitter than glutamate (such as cholinergic striatal interneurons and 5-HT neurons) express VGLUT3. It was recently reported that VGLUT3 increases acetylcholine vesicular filling, thereby, stimulating cholinergic transmission. This new regulatory mechanism is herein designated as vesicular-filling synergy (or vesicular synergy). In the present report, we found that deletion of VGLUT3 increased several anxiety-related behaviors in adult and in newborn mice as early as 8 d after birth. This precocious involvement of a vesicular glutamate transporter in anxiety led us to examine the underlying functional implications of VGLUT3 in 5-HT neurons. On one hand, VGLUT3 deletion caused a significant decrease of 5-HT(1A)-mediated neurotransmission in raphe nuclei. On the other hand, VGLUT3 positively modulated 5-HT transmission of a specific subset of 5-HT terminals from the hippocampus and the cerebral cortex. VGLUT3- and VMAT2-positive serotonergic fibers show little or no 5-HT reuptake transporter. These results unravel the existence of a novel subset of 5-HT terminals in limbic areas that might play a crucial role in anxiety-like behaviors. In summary, VGLUT3 accelerates 5-HT transmission at the level of specific 5-HT terminals and can exert an inhibitory control at the raphe level. Furthermore, our results suggest that the loss of VGLUT3 expression leads to anxiety-associated behaviors and should be considered as a potential new target for the treatment of this disorder.

The vesicular glutamate transporter VGLUT3 synergizes striatal acetylcholine tone.

Three subtypes of vesicular transporters accumulate glutamate into synaptic vesicles to promote its vesicular release. One of the subtypes, VGLUT3, is expressed in neurons, including cholinergic striatal interneurons, that are known to release other classical transmitters. Here we showed that disruption of the Slc17a8 gene (also known as Vglut3) caused an unexpected hypocholinergic striatal phenotype. Vglut3(-/-) mice were more responsive to cocaine and less prone to haloperidol-induced catalepsy than wild-type littermates, and acetylcholine release was decreased in striatum slices lacking VGLUT3. These phenotypes were associated with a colocalization of VGLUT3 and the vesicular acetylcholine transporter (VAChT) in striatal synaptic vesicles and the loss of a synergistic effect of glutamate on vesicular acetylcholine uptake. We propose that this vesicular synergy between two transmitters is the result of the unbalanced bioenergetics of VAChT, which requires anion co-entry for continuing vesicular filling. Our study reveals a previously unknown effect of glutamate on cholinergic synapses with potential functional and pharmacological implications.

Loss of VGLUT1 and VGLUT2 in the prefrontal cortex is correlated with cognitive decline in Alzheimer disease.

Several lines of evidence suggest that the glutamatergic system is severely impaired in Alzheimer disease (AD). Here, we assessed the status of glutamatergic terminals in AD using the first available specific markers, the vesicular glutamate transporters VGLUT1 and VGLUT2. We quantified VGLUT1 and VGLUT2 in the prefrontal dorsolateral cortex (Brodmann area 9) of controls and AD patients using specific antiserums. A dramatic decrease in VGLUT1 and VGLUT2 was observed in AD using Western blot. Similar decreases were observed in an independent group of subjects using immunoautoradiography. The VGLUT1 reduction was highly correlated with the degree of cognitive impairment, assessed with the clinical dementia rating (CDR) score. A significant albeit weaker correlation was also observed with VGLUT2. These findings provide evidence indicating that glutamatergic systems are severely impaired in the A9 region of AD patients and that this impairment is strongly correlated with the progression of cognitive decline. Our results suggest that VGLUT1 expression in the prefrontal cortex could be used as a valuable neurochemical marker of dementia in AD.

Sex-dependent up-regulation of two splicing factors, Psf and Srp20, during hippocampal memory formation.

Gene transcription is required for long-term memory (LTM) formation. LTM formation is impaired in a male-specific manner in mice lacking either of the two Ca(2+)/calmodulin-dependent kinase kinase (Camkk) genes. Since altered transcription was suggested to cause these impairments in LTM formation, we used microarrays to screen for CaMKKbeta-dependent gene expression changes. Here we show that the hippocampal mRNA expression of two splicing factors, splicing factor arginine/serine-rich 3 (Sfrs3/Srp20) and polypyrimidine tract-binding protein-associated splicing factor (Psf), is altered in CaMKKbeta-deficient males. In wild-type (WT) mice, the basal expression level in the hippocampus is higher in males than in females, and the sex difference in Srp20 expression is detectable before puberty. Training in two hippocampus-dependent learning tasks, the spatial version of the Morris water maze (MWM) and background contextual fear conditioning, increases the hippocampal mRNA expression of both splicing factors in WT males. However, the increase in Srp20 mRNA expression occurs only in males and not in females, whereas the up-regulation of Psf expression occurs in both sexes. Importantly, control experiments demonstrate that the up-regulation of both splicing factors is specific for the learned associations after contextual fear conditioning. In summary, we provide the first evidence for a regulation of splicing factors during LTM formation and we suggest that alternative splicing contributes to sex differences in LTM formation.

Anxiety in mice: a principal component analysis study.

Two principal component analyses of anxiety were undertaken investigating two strains of mice (ABP/Le and C57BL/6ByJ) in two different experiments, both classical tests for assessing anxiety in rodents. The elevated plus-maze and staircase were used for the first experiment, and a free exploratory paradigm and light-dark discrimination were used for the second. The components in the analyses produced definitions of four fundamental behavior patterns: novelty-induced anxiety, general activity, exploratory behavior, and decision making. We also noted that the anxious phenotype was determined by both strain and experimental procedure. The relationship between behavior patterns and the use of specific tests plus links with the genetic background are discussed.

An endogenous inhibitor of calcium/calmodulin-dependent kinase II is up-regulated during consolidation of fear memory.

CaMKIINalpha and CaMKIINbeta are endogenous inhibitors of the abundant synaptic protein, calcium/calmodulin-dependent protein kinase II (CaMKII). CaMKII exerts a prominent function in memory formation and the endogenous inhibitors might be important regulators of CaMKII activity during this process. Here we investigated whether or not CaMKIINalpha and CaMKIINbeta gene expressions are regulated in the mouse hippocampus and amygdala after background contextual fear conditioning. Quantitative real-time PCR revealed that the hippocampal expression of CaMKIINalpha mRNA was up-regulated 30 and 60 min after conditioning. In contrast, CaMKIINbeta mRNA expression did not change. The up-regulation of CaMKIINalpha expression was specific for the fear memory because the context alone and a shock control did not induce any variation of transcription level. Quantification of in situ hybridization signals showed that CaMKIINalpha expression increased in hippocampal area CA1, in the dentate gyrus (DG) and in the lateral amygdala (LA) 30 min after training. Our findings show an up-regulation in the expression of the endogenous inhibitor gene CaMKIINalpha during consolidation of fear memory. The early onset and the amplitude of the up-regulation are similar to those of immediate-early genes. Taken together, our results suggest that the CaMKIINalpha inhibitor has a physiological role in controlling CaMKII activity from an early stage of memory consolidation.

Spatio-temporal analysis of locomotion in BALB/cByJ and C57BL/6J mice in different environmental conditions.

We analysed spatial and temporal characteristics of mouse locomotion and investigated whether mouse gait differed between strains and environments. To this end, we used two inbred strains of mice (BALB/cByJ and C57BL/6J) known for their contrasting inherent level of anxiety, in three different visual surroundings. The animal position was determined relative to each environment. Gait cycle, defined as the sequence of limb movements, was analysed relative to time. We also recorded spatial parameters of gait such as stride length, track width and footfall characteristics. These measures allowed us to obtain an accurate description of locomotion and to assess subtle modifications of the gait. We found that mice adjusted their position in space, posture and gait in order to either stabilize their body on the ground in a potentially unsafe environment, or to optimize propulsion and maneuverability in a safer location. In addition, the two strains of mice used different strategies, suggesting that the inherent level of emotionality may affect the organization of locomotion in mice. We further concluded that higher structures of the central nervous system are involved in the online control of locomotion.

Posture and balance responses to a sensory challenge are related to anxiety in mice.

Anxiety disorders and balance disorders share common clinical features related to perception such as spatial disorientation or dizziness. The search for the mechanism underlying this core of symptoms led us to investigate impairments in multisensory integration. In mice, the 'rotating beam test' allows analysis of changes in balance control and posture in response to a multisensory challenge. We used the BALB/c and C57BL/6 inbred strains of mice, known for their contrasted anxiety-related behavior. The level of anxiety was also manipulated using anxiolytic and anxiogenic pharmacological compounds. Despite equal sensori-motor abilities, anxious mice were more prone to fall off the rotating beam and showed more imbalance than non-anxious mice. Striking inter-strain differences in posture were also observed. Diazepam and beta-CCM reversed these strain-specific responses in opposite directions. We demonstrated that balance and postural strategies developed in response to a multisensory challenge vary as a function of the level of anxiety in mice.