Circadian patterns of behaviour change during pregnancy in mice.

Food intake and activity adapt during pregnancy to meet the increased energy demands. In comparison to non-pregnant females, pregnant mice consume more food, eating larger meals during the light phase, and reduce physical activity. How pregnancy changes the circadian timing of behaviour was less clear. We therefore randomised female C57BL/6J mice to mating for study until early (n = 10), mid- (n = 10) or late pregnancy (n = 11) or as age-matched, non-pregnant controls (n = 12). Mice were housed individually in Promethion cages with a 12 h light-12 h dark cycle [lights on at 07.00 h, Zeitgeber (ZT)0] for behavioural analysis. Food intake between ZT10 and ZT11 was greater in pregnant than non-pregnant mice on days 6.5-12.5 and 12.5-17.5. In mice that exhibited a peak in the last 4 h of the light phase (ZT8-ZT12), peaks were delayed by 1.6 h in the pregnant compared with the non-pregnant group. Food intake immediately after dark-phase onset (ZT13-ZT14) was greater in the pregnant than non-pregnant group during days 12.5-17.5. Water intake patterns corresponded to food intake. From days 0.5-6.5 onwards, the pregnant group moved less during the dark phase, with decreased probability of being awake, in comparison to the non-pregnant group. The onset of dark-phase activity, peaks in activity, and wakefulness were all delayed during pregnancy. In conclusion, increased food intake during pregnancy reflects increased amplitude of eating behaviour, without longer duration. Decreases in activity also contribute to positive energy balance in pregnancy, with delays to all measured behaviours evident from mid-pregnancy onwards. KEY POINTS: Circadian rhythms synchronise daily behaviours including eating, drinking and sleep, but how these change in pregnancy is unclear. Food intake increased, with delays in peaks of food intake behaviour late in the light phase from days 6.5 to 12.5 of pregnancy, in comparison to the non-pregnant group. The onset of activity after lights off (dark phase) was delayed in pregnant compared with non-pregnant mice. Activity decreased by ∼70% in the pregnant group, particularly in the dark (active) phase, with delays in peaks of wakefulness evident from days 0.5-6.5 of pregnancy onwards. These behavioural changes contribute to positive energy balance during pregnancy. Delays in circadian behaviours during mouse pregnancy were time period and pregnancy stage specific, implying different regulatory mechanisms.

Plasticity of gastrointestinal vagal afferents in terms of feeding-related physiology and pathophysiology.

Gastrointestinal vagal afferents play an important role in communicating food related information from the gut to the brain. This information initiates vago-vagal reflexes essential for gut functions, including gut motility and secretions. These afferents also play a role in energy homeostasis, signalling the arrival, amount and nutrient composition of a meal to the central nervous system where it is processed ultimately leading to termination of a meal. Vagal afferent responses to food related stimuli demonstrate a high degree of plasticity, responding to short term changes in nutritional demand, such as the fluctuations that occur across a 24-hr or in response to a fast, as well as long term changes in energy demand, such as occurs during pregnancy. This plasticity is disrupted in disease states, such as obesity or chronic stress where there is hypo- and hypersensitivity of these afferents, respectively. Improved understanding of the plasticity of these afferents will enable identification of new treatment options for diseases associated with vagal afferent function.

Advancing translational research for colorectal immuno-oncology.

Colorectal cancer (CRC) is a common and deadly disease. Unfortunately, immune checkpoint inhibitors (ICIs) fail to elicit effective anti-tumour responses in the vast majority of CRC patients. Patients that are most likely to respond are those with DNA mismatch repair deficient (dMMR) and microsatellite instability (MSI) disease. However, reliable predictors of ICI response are lacking, even within the dMMR/MSI subtype. This, together with identification of novel mechanisms to increase response rates and prevent resistance, are ongoing and vitally important unmet needs. To address the current challenges with translation of early research findings into effective therapeutic strategies, this review summarises the present state of preclinical testing used to inform the development of immuno-regulatory treatment strategies for CRC. The shortfalls and advantages of commonly utilised mouse models of CRC, including chemically induced, transplant and transgenic approaches are highlighted. Appropriate use of existing models, incorporation of patient-derived data and development of cutting-edge models that recapitulate important features of human disease will be key to accelerating clinically relevant research in this area.

Active glucose transport varies by small intestinal region and oestrous cycle stage in mice.

NEW FINDINGS: What is the central question of this study? Body mass and food intake change during the female ovarian cycle: does glucose transport by the small intestine also vary? What is the main finding and its importance? We have optimised Ussing chamber methodology to measure region-specific active glucose transport in the small intestine of adult C57BL/6 mice. Our study provides the first evidence that jejunal active glucose transport changes during the oestrous cycle in mice, and is higher at pro-oestrus than oestrus. These results demonstrate adaptation in active glucose uptake, concurrent with previously reported changes in food intake. ABSTRACT: Food intake changes across the ovarian cycle in rodents and humans, with a nadir during the pre-ovulatory phase and a peak during the luteal phase. However, it is unknown whether the rate of intestinal glucose absorption also changes. We therefore mounted small intestinal sections from C57BL/6 female mice (8-9 weeks old) in Ussing chambers and measured active ex vivo glucose transport via the change in short-circuit current (∆Isc ) induced by glucose. Tissue viability was confirmed by a positive ∆Isc response to 100 µM carbachol following each experiment. Active glucose transport, assessed after addition of 5, 10, 25 or 45 mM d-glucose to the mucosal chamber, was highest at 45 mM glucose in the distal jejunum compared to duodenum and ileum (P < 0.01). Incubation with the sodium-glucose cotransporter 1 (SGLT1) inhibitor phlorizin reduced active glucose transport in a dose-dependent manner in all regions (P < 0.01). Active glucose uptake induced by addition of 45 mM glucose to the mucosal chamber in the absence or presence of phlorizin was assessed in jejunum at each oestrous cycle stage (n = 9-10 mice per stage). Overall, active glucose uptake was lower at oestrus compared to pro-oestrus (P = 0.025). This study establishes an ex vivo method to measure region-specific glucose transport in the mouse small intestine. Our results provide the first direct evidence that SGLT1-mediated glucose transport in the jejunum changes across the ovarian cycle. The mechanisms underlying these adaptations in nutrient absorption remain to be elucidated.

The association between diet quality, plant-based diets, systemic inflammation, and mortality risk: findings from NHANES.

PURPOSE: To our knowledge, no studies have examined the association of diet quality and plant-based diets (PBD) with inflammatory-related mortality in obesity. Therefore, this study aimed to determine the joint associations of Healthy Eating Index-2015 (HEI-2015), plant-based dietary index (PDI), healthy PDI (hPDI), unhealthy PDI (uPDI), pro-vegetarian dietary index (PVD), and systemic inflammation with all-cause, cardiovascular disease (CVD), and cancer mortality risks by obesity status. METHODS: Participants from NHANES were included in cross-sectional (N = 27,915, cycle 1999-2010, 2015-2018) and longitudinal analysis (N = 11,939, cycle 1999-2008). HEI-2015, PDI, hPDI, uPDI, and PVD were constructed based on the 24-h recall dietary interview. The grade of inflammation (low, moderate, and high) was determined based on C-reactive protein (CRP) values and multivariable ordinal logistic regression was used to determine the association. Cox proportional hazard models were used to determine the joint associations of diet and inflammation with mortality. RESULTS: In the fully adjusted model, HEI-2015 (ORT3vsT1 = 0.76, 95% CI 0.69-0.84; p-trend = < 0.001), PDI (ORT3vsT1 = 0.83, 95% CI 0.75-0.91; p trend = < 0.001), hPDI (ORT3vsT1 = 0.79, 95% CI 0.71-0.88; p trend = < 0.001), and PVD (ORT3vsT1 = 0.85, 95% CI 0.75-0.97; p trend = 0.02) were associated with lower systemic inflammation. In contrast, uPDI was associated with higher systemic inflammation (ORT3vsT1 = 1.18, 95% CI 1.06-1.31; p-trend = 0.03). Severe inflammation was associated with a 25% increase in all-cause mortality (ORT3vsT1 = 1.25, 95% CI 1.03-1.53, p trend = 0.02). No association was found between PDI, hPDI, uPDI, and PVD with mortality. The joint association, between HEI-2015, levels of systemic inflammation, and all-cause, CVD and cancer mortality, was not significant. However, a greater reduction in mortality risk with an increase in HEI-2015 scores was observed in individuals with low and moderate inflammation, especially those with obesity. CONCLUSION: Higher scores of HEI-2015 and increased intake of a healthy plant-based diet were associated with lower inflammation, while an unhealthy plant-based diet was associated with higher inflammation. A greater adherence to the 2015 dietary guidelines may reduce the risk of mortality associated with inflammation and may also benefit individuals with obesity who had low and moderate inflammation.

The gut microbiome regulates host glucose homeostasis via peripheral serotonin.

The gut microbiome is an established regulator of aspects of host metabolism, such as glucose handling. Despite the known impacts of the gut microbiota on host glucose homeostasis, the underlying mechanisms are unknown. The gut microbiome is also a potent mediator of gut-derived serotonin synthesis, and this peripheral source of serotonin is itself a regulator of glucose homeostasis. Here, we determined whether the gut microbiome influences glucose homeostasis through effects on gut-derived serotonin. Using both pharmacological inhibition and genetic deletion of gut-derived serotonin synthesis, we find that the improvements in host glucose handling caused by antibiotic-induced changes in microbiota composition are dependent on the synthesis of peripheral serotonin.

Pregnancy-related plasticity of gastric vagal afferent signals in mice.

Gastric vagal afferents (GVAs) sense food-related mechanical stimuli and signal to the central nervous system, to integrate control of meal termination. Pregnancy is characterized by increased maternal food intake, which is essential for normal fetal growth and to maximize progeny survival and health. However, it is unknown whether GVA function is altered during pregnancy to promote food intake. This study aimed to determine the mechanosensitivity of GVAs and food intake during early, mid-, and late stages of pregnancy in mice. Pregnant mice consumed more food compared with nonpregnant mice, notably in the light phase during mid- and late pregnancy. The increased food intake was predominantly due to light-phase increases in meal size across all stages of pregnancy. The sensitivity of GVA tension receptors to gastric distension was significantly attenuated in mid- and late pregnancy, whereas the sensitivity of GVA mucosal receptors to mucosal stroking was unchanged during pregnancy. To determine whether pregnancy-associated hormonal changes drive these adaptations, the effects of estradiol, progesterone, prolactin, and growth hormone on GVA tension receptor mechanosensitivity were determined in nonpregnant female mice. The sensitivity of GVA tension receptors to gastric distension was augmented by estradiol, attenuated by growth hormone, and unaffected by progesterone or prolactin. Together, the data indicate that the sensitivity of GVA tension receptors to tension is reduced during pregnancy, which may attenuate the perception of gastric fullness and explain increased food intake. Further, these adaptations may be driven by increases in maternal circulating growth hormone levels during pregnancy.NEW & NOTEWORTHY This study provides first evidence that gastric vagal afferent signaling is attenuated during pregnancy and inversely associated with meal size. Growth hormone attenuated mechanosensitivity of gastric vagal afferents, adding support that increases in maternal growth hormone may mediate adaptations in gastric vagal afferent signaling during pregnancy. These findings have important implications for the peripheral control of food intake during pregnancy.

Leptin signaling in vagal afferent neurons supports the absorption and storage of nutrients from high-fat diet.

OBJECTIVE: Activation of vagal afferent neurons (VAN) by postprandial gastrointestinal signals terminates feeding and facilitates nutrient digestion and absorption. Leptin modulates responsiveness of VAN to meal-related gastrointestinal signals. Rodents with high-fat diet (HF) feeding develop leptin resistance that impairs responsiveness of VAN. We hypothesized that lack of leptin signaling in VAN reduces responses to meal-related signals, which in turn decreases absorption of nutrients and energy storage from high-fat, calorically dense food. METHODS: Mice with conditional deletion of the leptin receptor from VAN (Nav1.8-Cre/LepRfl/fl; KO) were used in this study. Six-week-old male mice were fed a 45% HF for 4 weeks; metabolic phenotype, food intake, and energy expenditure were measured. Absorption and storage of nutrients were investigated in the refed state. RESULTS: After 4 weeks of HF feeding, KO mice gained less body weight and fat mass that WT controls, but this was not due to differences in food intake or energy expenditure. KO mice had reduced expression of carbohydrate transporters and absorption of carbohydrate in the jejunum. KO mice had fewer hepatic lipid droplets and decreased expression of de novo lipogenesis-associated enzymes and lipoproteins for endogenous lipoprotein pathway in liver, suggesting decreased long-term storage of carbohydrate in KO mice. CONCLUSIONS: Impairment of leptin signaling in VAN reduces responsiveness to gastrointestinal signals, which reduces intestinal absorption of carbohydrates and de novo lipogenesis resulting in reduced long-term energy storage. This study reveals a novel role of vagal afferents to support digestion and energy storage that may contribute to the effectiveness of vagal blockade to induce weight loss.

Nutrient patterns and depressive symptoms among Australian adults.

PURPOSE: Much of the current literature on the associations between diet and depression focus on single nutrients rather than nutrient patterns (NPs). We investigated the association between NPs and depressive symptoms (DepS) in an Australian adult population. METHODS: DepS were examined at two different time points, in 2010 (Stage 3, n = 1743, 49.0% males) and 2015 [North West 2015 (NW15), n = 1,024, 46.6% males] of the North West Adelaide Health Study (NWAHS). Dietary habits were evaluated using a food frequency questionnaire (FFQ) at Stage 3. DepS were assessed using the Center for Epidemiological Studies Depression (CES-D) scale at Stage 3 and NW15. Principal component analysis was used to identify NPs as well as the factor structure of the CES-D. Log- and negative binomial regression analyses were used to assess the association between NPs and DepS scores. Ordinal logistic regression analysis was undertaken between the NPs and identified factors of the CES-D score. RESULTS: Three NPs (from the FFQ) and two-factors (from the CES-D score) were obtained. After adjusting for known confounding variables, a 'plant-sourced' NP (ß-carotene, fibre, vitamin C, potassium and α-carotene) was inversely associated with DepS at Stage 3 [prevalence ratio (PR)Q4VsQ1, 0.78; 95% CI 0.66-0.92; p = 0.003], whereas an 'animal-sourced' (ω-3 fatty acid, monounsaturated fat, vitamin E and cholesterol) or 'mixed-source' (phosphorous, protein, vitamin B2, iodine and zinc) NP was not associated with DepS. There was an inverse relationship between the 'plant-sourced' NP and the '(absence of) positive-affect' factor from the CES-D in both stages. CONCLUSION: The 'plant-sourced' NP is consistently and inversely associated with DepS; however, longitudinal studies are recommended to confirm these results.

Role of Indices Incorporating Power, Force and Time in AF Ablation: A Systematic Review of Literature.

INTRODUCTION: Successful pulmonary vein isolation (PVI) for atrial fibrillation (AF) depends on the formation of durable transmural lesions. Recently, novel indices have emerged to guide lesion delivery. The aim of the systematic review of literature was to determine AF recurrence following ablation guided by indices incorporating force, power and time, and compare acute procedural outcomes and 12-month AF recurrence with ablation guided by contact force (CF) guided only. METHODS: PubMed, EMBASE, and Web of Science Core Collection databases were searched on 27 January 2020 using the keywords; catheter ablation, ablation index (AI), lesion size index (LSI), contact force, atrial fibrillation. RESULTS: After exclusions, seven studies were included in the analysis. AI-guided catheter ablation was associated with a 91% (n=5, 0.91 95% CI; 0.88-0.93) and 80% (n=5, 0.80, 95% CI; 0.77-0.84) freedom from AF at 12 months with and without the use of anti-arhythmic drugs respectively. As compared to CF guided ablation, AI-guided catheter ablation was associated with a 49% increase in successful first pass isolation (n=3; RR: 1.49, 95% CI; 1.38, 1.61), a 50% decrease in number of acute reconnections (n=4; RR: 0.50, 95% CI; 0.39-0.65) and a 22% (n=4, RR: 1.22, 95% CI; 1.10-1.35) increase in AF freedom without anti-arrhythmic drugs at 12 months. CONCLUSIONS: Radiofrequency ablation guided by AI was associated with higher successful first pass isolation and lower rates of acute reconnection which translates to greater freedom from AF at 12 months [CRD42019131469].

Modulatory effect of methanandamide on gastric vagal afferent satiety signals depends on nutritional status.

SIGNIFICANCE STATEMENT: Gastric vagal afferent responses to tension are dampened in high fat diet-induced obesity. Endocannabinoids are known to dose-dependently inhibit and excite gastric vagal afferents but their effect on gastric vagal afferents in diet-induced obesity are unknown. In individual gastric vagal afferent neurons of diet-induced obese mice the co-expression of components of the endocannabinoid system, including CB1, GHSR, TRPV1 and FAAH, was increased compared with lean mice. In high fat diet-induced obese mice, methanandamide only inhibited gastric vagal afferent responses to tension, possibly due to the observed change in the balance of receptors, hormones and breakdown enzymes in this system. Collectively, these data suggest that endocannabinoid signalling, by gastric vagal afferents, is altered in diet-induced obesity which may impact satiety and gastrointestinal function. ABSTRACT: Gastric vagal afferents (GVAs) play a role in appetite regulation. The endocannabinoid anandamide (AEA) dose-dependently inhibits and excites tension-sensitive GVAs. However, it is also known that high fat diet (HFD) feeding alters GVA responses to stretch. The aim of this study was to determine the role of AEA in GVA signalling in lean and HFD-induced obese mice. Male C57BL/6 mice were fed (12 weeks) a standard laboratory diet (SLD) or HFD. Protein and mRNA expression of components of the cannabinoid system was determined in individual GVA cell bodies and the gastric mucosa. An in vitro GVA preparation was used to assess the effect of methanandamide (mAEA) on tension-sensitive GVAs and the second messenger pathways involved. In individual GVA cell bodies, cannabinoid 1 (CB1) and ghrelin (GHSR) receptor mRNA was higher in HFD mice than SLD mice. Conversely, gastric mucosal AEA and ghrelin protein levels were lower in HFD mice than SLD mice. In SLD mice, mAEA exerted dose-dependent inhibitory and excitatory effects on tension-sensitive GVAs. Only an inhibitory effect of mAEA was observed in HFD mice. The excitatory effect of mAEA was dependent on CB1, transient receptor potential vanilloid 1 (TRPV1) and the protein kinase C. Conversely, the inhibitory effect was dependent on CB1, growth hormone secretagogue receptor, TRPV1 and the protein kinase A. Endocannabinoids, acting through CB1 and TRPV1, have a pivotal role in modulating GVA satiety signals depending on the second messenger pathway utilised. In HFD mice only an inhibitory effect was observed. These changes may contribute to the development and/or maintenance of obesity.

Biphasic effects of methanandamide on murine gastric vagal afferent mechanosensitivity.

KEY POINTS: The fine control of food intake is important for the maintenance of a healthy metabolic state. Gastric vagal afferents (GVAs) are involved in the peripheral regulation of food intake via signalling the degree of distension of the stomach which ultimately leads to feelings of fullness and satiety. This study provides evidence that endocannabinoids such as anandamide are capable of regulating GVA sensitivity in a concentration-dependent biphasic manner. This biphasic effect is dependent upon interactions between the CB1, TRPV1 and GHSR receptors. These data have important implications for the peripheral control of food intake. ABSTRACT: Gastric vagal afferents (GVAs) signal to the hindbrain resulting in satiety. Endocannabinoids are endogenous ligands of cannabinoid 1 receptor (CB1) and transient receptor potential vanilloid-1 (TRPV1) channels. The endocannabinoid anandamide (AEA) is expressed in the stomach, and its receptor CB1 is expressed in ghrelin-positive gastric mucosal cells. Further, TRPV1, CB1 and growth hormone secretagogue receptor (ghrelin receptor, GHSR) are expressed in subpopulations of GVA neurons. This study aimed to determine the interaction between TRPV1, CB1, GHSR and endocannabinoids in the modulation of GVA signalling. An in vitro electrophysiology preparation was used to assess GVA mechanosensitivity in male C57BL/6 mice. Effects of methanandamide (mAEA; 1-100 nm), on GVA responses to stretch were determined in the absence and presence of antagonists of CB1, TRPV1, GHSR, protein kinase-A (PKA), protein kinase-C (PKC) and G-protein subunits Gαi/o , or Gαq . Low doses (1-10 nm) of mAEA reduced GVA responses to 3 g stretch, whereas high doses (30-100 nm) increased the response. The inhibitory and excitatory effects of mAEA (1-100 nm) were reduced/lost in the presence of a CB1 and TRPV1 antagonist. PKA, Gαi/o or GHSR antagonists prevented the inhibitory effect of mAEA on GVA mechanosensitivity. Conversely, in the presence of a PKC or Gαq antagonist the excitatory effect of mAEA was reduced or lost, respectively. Activation of CB1, by mAEA, can activate or inhibit TRPV1 to increase or decrease GVA responses to stretch, depending on the pathway activated. These interactions could play an important role in the fine control of food intake.

Time-Restricted Feeding Prevents Ablation of Diurnal Rhythms in Gastric Vagal Afferent Mechanosensitivity Observed in High-Fat Diet-Induced Obese Mice.

Mechanosensitive gastric vagal afferents (GVAs) are involved in the regulation of food intake. GVAs exhibit diurnal rhythmicity in their response to food-related stimuli, allowing time of day-specific satiety signaling. This diurnal rhythmicity is ablated in high-fat-diet (HFD)-induced obesity. Time-restricted feeding (TRF) has a strong influence on peripheral clocks. This study aimed to determine whether diurnal patterns in GVA mechanosensitivity are entrained by TRF. Eight-week-old male C57BL/6 mice (N = 256) were fed a standard laboratory diet (SLD) or HFD for 12 weeks. After 4 weeks of diet acclimatization, the mice were fed either ad libitum or only during the light phase [Zeitgeber time (ZT) 0-12] or dark phase (ZT12-24) for 8 weeks. A subgroup of mice from all conditions (n = 8/condition) were placed in metabolic cages. After 12 weeks, ex vivo GVA recordings were taken at 3 h intervals starting at ZT0. HFD mice gained more weight than SLD mice. TRF did not affect weight gain in the SLD mice, but decreased weight gain in the HFD mice regardless of the TRF period. In SLD mice, diurnal rhythms in food intake were inversely associated with diurnal rhythmicity of GVA mechanosensitivity. These diurnal rhythms were entrained by the timing of food intake. In HFD mice, diurnal rhythms in food intake and diurnal rhythmicity of GVA mechanosensitivity were dampened. Loss of diurnal rhythmicity in HFD mice was abrogated by TRF. In conclusion, diurnal rhythmicity in GVA responses to food-related stimuli can be entrained by food intake. TRF prevents the loss of diurnal rhythmicity that occurs in HFD-induced obesity.SIGNIFICANCE STATEMENT Diurnal control of food intake is vital for maintaining metabolic health. Diet-induced obesity is associated with strong diurnal changes in food intake. Vagal afferents are involved in regulation of feeding behavior, particularly meal size, and exhibit diurnal fluctuations in mechanosensitivity. These diurnal fluctuations in vagal afferent mechanosensitivity are lost in diet-induced obesity. This study provides evidence that time-restricted feeding entrains diurnal rhythmicity in vagal afferent mechanosensitivity in lean and high-fat-diet (HFD)-induced obese mice and, more importantly, prevents the loss of rhythmicity in HFD-induced obesity. These data have important implications for the development of strategies to treat obesity.

A novel role for the extracellular matrix glycoprotein-Tenascin-X in gastric function.

KEY POINTS: Tenascin X (TNX) functions in the extracellular matrix of skin and joints where it maintains correct intercellular connections and tissue architecture TNX is associated exclusively with vagal-afferent endings and some myenteric neurones in mouse and human stomach, respectively. TNX-deficient mice have accelerated gastric emptying and hypersensitivity of gastric vagal mechanoreceptors that can be normalized by an inhibitor of vagal-afferent sensitivity. Cultured nodose ganglion neurones showed no changes in response to capsaicin, cholecystokinin and potassium chloride in TNX-deficient mice. TNX-deficient patients have upper gastric dysfunction consistent with those in a mouse model. Our translational studies suggest that abnormal gastric sensory function may explain the upper gut symptoms present in TNX deficient patients, thus making it important to study gastric physiology. TNX deficiency should be evaluated routinely in patients with connective tissue abnormalities, which will enable a better understanding of its role and allow targeted treatment. For example, inhibitors of vagal afferents-baclofen could be beneficial in patients. These hypotheses need confirmation via targeted clinical trials. ABSTRACT: Tenascin-X (TNX) is a glycoprotein that regulates tissue structure via anti-adhesive interactions with collagen in the extracellular matrix. TNX deficiency causes a phenotype similar to hypermobility Ehlers-Danlos syndrome involving joint hypermobility, skin hyperelasticity, pain and gastrointestinal dysfunction. Previously, we have shown that TNX is required for neural control of the bowel by a specific subtype of mainly cholinergic enteric neurones and regulates sprouting and sensitivity of nociceptive sensory endings in mouse colon. These findings correlate with symptoms shown by TNX-deficient patients and mice. We aimed to identify whether TNX is similarly present in neural structures found in mouse and human gastric tissue. We then determined whether TNX has a functional role, specifically in gastric motor and sensory function and nodose ganglia neurones. We report that TNX was present in calretinin-immunoreactive extrinsic nerve endings in mouse and human stomach. TNX deficient mice had accelerated gastric emptying and markedly increased vagal afferent responses to gastric distension that could be rescued with GABAB receptor agonist. There were no changes in nodose ganglia excitability in TNX deficient mice, suggesting that vagal afferent responses are probably the result of altered peripheral mechanosensitivity. In TNXB-deficient patients, significantly greater symptoms of reflux, indigestion and abdominal pain were reported. In the present study, we report the first role for TNX in gastric function. Further studies are required in TNX deficient patients to determine whether symptoms can be relieved using GABAB agonists.

The TRPV1 channel regulates glucose metabolism.

Endocannabinoids (ECs) mediate effects via cannabinoid receptor types 1 and 2 (CB1 and 2) and transient receptor potential channel-vanilloid subfamily member 1 (TRPV1) channels. In high-fat diet (HFD)-induced obese mice overactivity of the EC system and inhibition of CB1 increase skeletal muscle glucose uptake. We explored the role of TRPV1. Male TRPV1+/+(WT) and TRPV1-/-(KO)-mice were fed (20 wk) a standard laboratory diet (SLD) or HFD. An intraperitoneal glucose tolerance test was performed. RT-PCR was performed to measure mRNA of genes involved in glucose/lipid metabolism and the EC system in soleus (SOL) and extensor digitorum longus (EDL) muscles. Cultured L6 cells were used to measure glucose uptake in skeletal muscle. HFD mice weighed more and had higher insulin levels than SLD mice, with no genotype differences. Basal and peak glucose were higher in HFD mice irrespective of genotype, but glucose cleared faster in HFD WT vs. HFD KO-mice. 2-Arachidonoylglycerol augmented insulin-induced glucose uptake in skeletal L6-cells, an effect blocked by the TRPV1 antagonist SB-366791. In EDL, fatty acid amide hydrolase (FAAH) mRNA was increased in KO vs. WT mice, irrespective of diet. Pyruvate dehydrogenase kinase isozyme 4 (PDK4) and mitochondrial uncoupling protein 3 (UCP3) were elevated and FA desaturase 2 (FADS2) mRNA lower in HFD mice, irrespective of genotype. CB1 and stearoyl-CoA desaturase 1 (SCD1) were lower in HFD WT mice only. In SOL, PDK4, UCP3, hormone-sensitive lipase (LIPE), fatty acid translocase (CD36), and carnitine palmitoyl transferase 2 (CPT2) were elevated and SCD1, FAAH, FADS2, and Troponin 1 (TNNC1) mRNA lower in HFD mice, irrespective of genotype. In conclusion, TRPV1 regulates glucose disposal in HFD mice. We propose that TRPV1 plays a role in coordinating glucose metabolism in EDL under conditions of metabolic stress.

Activation of CRF2 receptor increases gastric vagal afferent mechanosensitivity.

Gastric vagal afferent (GVA) sensing of food-related mechanical stimuli is a crucial mechanism in the control of feeding behavior and gastric function. Stress is an important factor contributing to eating disorders and gastric diseases. Chronic stress has been shown to increase the mechanosensitivity of GVAs in mice and to reduce food intake and body weight. Whether the mechanosensitivity of GVAs is modulated by stress hormones is not known. This study aimed to determine the effect of stress hormones on GVA mechanosensitivity. The expression of stress hormone receptors in GVA cell bodies was determined in 8-wk-old male C57BL/6 mice using quantitative RT-PCR combined with laser capture microdissection. The mechanosensitivity of GVAs was determined in the absence and presence of stress hormones using an in vitro single-fiber recording preparation. NR3C1 and CRHR2 (mRNA isoforms of glucocorticoid receptor and CRF2 receptor, respectively) were expressed in GVA neurons. The glucocorticoid receptor agonist corticosterone had no effect on the mechanosensitivity of either tension or mucosal GVAs. Activation of CRF2 receptor by its specific analog, urocortin 3, significantly increased the mechanosensitivity of both tension and mucosal GVAs, an effect prevented by the CRF2 receptor antagonist astressin 2B. In conclusion, activation of CRF2 receptor increases the mechanosensitivity of GVAs. This may contribute to the stress- and CRF2 receptor-associated changes in feeding behavior and gastric function, possibly contributing to the hypersensitivity of GVAs in chronic stress conditions.NEW & NOTEWORTHY Gastric vagal afferents (GVAs) relay food-related signals to the central nervous system, where they are processed, eventually leading to modulation of food intake and gastric function. GVA signaling can be modulated by an array of hormones. Stress has been shown to induce GVA hypersensitivity. This study demonstrates that GVA neurons express subtypes of stress hormone receptors, specifically CRF2. Furthermore, activation of CRF2 receptor increases GVA mechanosensitivity, which could have implications for food intake and gastric function.

A rotating light cycle promotes weight gain and hepatic lipid storage in mice.

Processes involved in regulation of energy balance and intermediary metabolism are aligned to the light-dark cycle. Shift-work and high-fat diet (HFD)-induced obesity disrupt circadian rhythmicity and are associated with increased risk of nonalcoholic fatty liver disease. This study aimed to determine the effect of simulating shift work on hepatic lipid accumulation in lean and HFD mice. C57BL/6 mice fed a standard laboratory diet (SLD) or HFD for 4 wk were further allocated to a normal light (NL) cycle (lights on: 0600-1800) or rotating light (RL) cycle [3 days NL and 4 days reversed (lights on: 1800-0600) repeated] for 8 wk. Tissue was collected every 3 h beginning at 0600. HFD mice gained more weight than SLD mice, and RL mice gained more weight than NL mice. SLD-NL and HFD-NL mice, but not RL mice, were more active, had higher respiratory quotients, and consumed/expended more energy during the dark phase compared with the light phase. Blood glucose and plasma cholesterol and triglyceride concentrations were elevated in HFD and SLD-RL compared with SLD-NL mice. Hepatic glycogen was elevated in HFD compared with SLD mice. Hepatic triglycerides were elevated in SLD-RL and HFD mice compared with SLD-NL. Circadian rhythmicity of hepatic acetyl-CoA carboxylase (ACACA) mRNA was phase shifted in SLD-RL and HFD-NL and lost in HFD-RL mice. Hepatic ACACA protein was reduced in SLD-RL and HFD mice compared with SLD-NL mice. Hepatic adipose triglyceride lipase was elevated in HFD-NL compared with SLD-NL but lower in RL mice compared with NL mice irrespective of diet. In conclusion, an RL cycle model of shift work promotes weight gain and hepatic lipid storage even in lean conditions. NEW & NOTEWORTHY In this publication we describe the effects of a rotating light cycle model of shift work in lean and high-fat diet-induced obese mice on body mass, diurnal patterns of energy intake and expenditure, and hepatic lipid storage. The data indicate that modeling shift work, via a rotating light cycle, promotes weight gain and hepatic lipid accumulation even in mice on a standard laboratory diet.

High-Fat Diet-Induced Obesity Ablates Gastric Vagal Afferent Circadian Rhythms.

Rats with high-fat diet (HFD)-induced obesity increase daytime eating, suggesting an alteration in circadian food intake mechanisms. Gastric vagal afferents (GVAs) respond to mechanical stimuli to initiate satiety. These signals are dampened in HFD mice and exhibit circadian variations inversely with food intake in lean mice. Furthermore, leptin shows circadian variation in its circulating level and is able to modulate GVA mechanosensitivity. However, whether leptin's ability to modulate GVAs occurs in a circadian manner is unknown. Therefore, we investigated whether changes in the circadian intake of food in HFD-induced obesity is associated with a disruption in GVA circadian rhythms. Eight-week-old male C57BL/6 mice were fed a standard laboratory diet (SLD) or a HFD for 12 weeks. A subgroup of SLD and HFD mice were housed in metabolic cages. After 12 weeks, ex vivo GVA recordings were taken at 3 h intervals starting at zeitgeber time 0 (ZT0) and stomach content was measured. After 12 weeks, HFD mice consumed more food during the light phase through larger and more frequent meals compared with SLD mice. SLD mice exhibited circadian fluctuation in stomach content, which peaked at ZT18 and reached a nadir at ZT9. At these time points, both tension and mucosal receptor mechanosensitivity were the lowest and highest, respectively. HFD mice exhibited little circadian variation in stomach content or GVA mechanosensitivity. Leptin potentiated mucosal receptor mechanosensitivity only in SLD mice and with reduced potency during the dark phase. In conclusion, loss of circadian variation in GVA signaling may underpin changes in eating behavior in HFD-induced obesity. SIGNIFICANCE STATEMENT: Appropriate circadian control of food intake is vital for maintaining metabolic health. Diet-induced obesity is associated with strong circadian changes in food intake, but the contributing mechanisms have yet to be determined. Vagal afferents are involved in regulation of feeding behavior, particularly meal size, and have been shown to exhibit circadian fluctuation in mechanosensitivity, potentially allowing for time of day-specific levels of satiety signaling. Our study indicates that, in diet-induced obesity, these circadian fluctuations in gastric vagal afferent mechanosensitivity are lost. This was accompanied by increased light phase eating, particularly increased meal size. This is the first evidence that diet-induced disruption to vagal afferent signaling may cause a perturbation in circadian eating patterns.

Mechanisms of activation of mouse and human enteroendocrine cells by nutrients.

OBJECTIVE: Inhibition of food intake and glucose homeostasis are both promoted when nutrients stimulate enteroendocrine cells (EEC) to release gut hormones. Several specific nutrient receptors may be located on EEC that respond to dietary sugars, amino acids and fatty acids. Bypass surgery for obesity and type II diabetes works by shunting nutrients to the distal gut, where it increases activation of nutrient receptors and mediator release, but cellular mechanisms of activation are largely unknown. We determined which nutrient receptors are expressed in which gut regions and in which cells in mouse and human, how they are associated with different types of EEC, how they are activated leading to hormone and 5-HT release. DESIGN AND RESULTS: mRNA expression of 17 nutrient receptors and EEC mediators was assessed by quantitative PCR and found throughout mouse and human gut epithelium. Many species similarities emerged, in particular the dense expression of several receptors in the distal gut. Immunolabelling showed specific colocalisation of receptors with EEC mediators PYY and GLP-1 (L-cells) or 5-HT (enterochromaffin cells). We exposed isolated proximal colonic mucosa to specific nutrients, which recruited signalling pathways within specific EEC extracellular receptor-regulated kinase (p-ERK) and calmodulin kinase II (pCAMKII), as shown by subsequent immunolabelling, and activated release of these mediators. Aromatic amino acids activated both pathways in mouse, but in humans they induced only pCAMKII, which was colocalised mainly with 5-HT expression. Activation was pertussis toxin-sensitive. Fatty acid (C12) potently activated p-ERK in human in all EEC types and evoked potent release of all three mediators. CONCLUSIONS: Specific nutrient receptors associate with distinct activation pathways within EEC. These may provide discrete, complementary pharmacological targets for intervention in obesity and type II diabetes.