Desynchrony between brain and peripheral clocks caused by CK1δ/ε disruption in GABA neurons does not lead to adverse metabolic outcomes.

Circadian disruption as a result of shift work is associated with adverse metabolic consequences. Internal desynchrony between the phase of the suprachiasmatic nuclei (SCN) and peripheral clocks is widely believed to be a major factor contributing to these adverse consequences, but this hypothesis has never been tested directly. A GABAergic Cre driver combined with conditional casein kinase mutations (Vgat-Cre+CK1δfl/flεfl/+ ) was used to lengthen the endogenous circadian period in GABAergic neurons, including the SCN, but not in peripheral tissues, to create a Discordant mouse model. These mice had a long (27.4 h) behavioral period to which peripheral clocks entrained in vivo, albeit with an advanced phase (∼6 h). Thus, in the absence of environmental timing cues, these mice had internal desynchrony between the SCN and peripheral clocks. Surprisingly, internal desynchrony did not result in obesity in this model. Instead, Discordant mice had reduced body mass compared with Cre-negative controls on regular chow and even when challenged with a high-fat diet. Similarly, internal desynchrony failed to induce glucose intolerance or disrupt body temperature and energy expenditure rhythms. Subsequently, a lighting cycle of 2-h light/23.5-h dark was used to create a similar internal desynchrony state in both genotypes. Under these conditions, Discordant mice maintained their lower body mass relative to controls, suggesting that internal desynchrony did not cause the lowered body mass. Overall, our results indicate that internal desynchrony does not necessarily lead to metabolic derangements and suggest that additional mechanisms contribute to the adverse metabolic consequences observed in circadian disruption protocols.

Behavioral thermoregulation in the fasted C57BL/6 mouse.

Under relatively cool ambient temperatures and a caloric deficit, mice will undergo daily torpor - a short-term regulated reduction in metabolic rate with a concomitant drop in body temperature. Mice can alternatively achieve metabolic savings by utilizing behavioral changes, such as seeking a warmer environment. However, there is a lack of knowledge about the behavioral interaction between torpor utilization and thermotaxis. That is, if a fasted mouse is faced with a choice between a warm environment not conducive for torpor, and a cool environment that will induce torpor, which scenario will the fasting mouse choose? Here, the temperature preferences of fasted mice were studied using a temperature gradient device that allows a mouse to freely move along a gradient of temperatures. C57BL/6 mice were implanted with temperature telemeters that recorded location, core temperature (Tb), and activity concurrently over a 23-h period in the thermal gradient. When the gradient was on, mice preferred the warm end of the gradient when fed (71 ± 4% of the time) and even more so when fasted (84 ± 2%). When the gradient was on, the fasted minimum Tb was significantly higher (34.4 ± 0.3 °C) than when the gradient was off (27.7 ± 1.6 °C). Further, fasted mice lost significantly more weight when the gradient was off despite maintenance of a metabolically favorable lower minimum Tb in this condition. These results indicate that fasted mice not only prefer warm ambient temperatures when given the choice, but that it is also the pathway with more favorable metabolic outcomes in a period of reduced caloric intake.

Physiological response to the odorant TMT in fully fed and calorically restricted laboratory mice.

2,3,5-trimethyl-3-thiazoline (TMT) is a chemical compound that is extracted from red fox urine and can be used to artificially simulate the presence of a predator. The purpose of this study was to test the hypothesis that TMT would block entry into torpor in the calorically restricted C57Bl/6 mouse. We first demonstrated that TMT induced fear in the mouse. Exposure to TMT induced an acute freeze response (67.2 ± 6.7% of time), as compared to 6.7 ± 1.7% when exposed to water. Further, exposure to TMT for 30 min led to elevated circulating corticosterone levels, 377 ± 33 ng/ml, as compared to 29 ± 4 ng/ml when exposed to water. When mice were exposed to TMT during the dark or light phase, body temperature (Tb) dropped by 1.7 ± 0.9 °C and 0.7 ± 1.1 °C, respectively, over the first 110 min after exposure. To determine whether TMT influences daily torpor, mice were calorically restricted and exposed to either water or TMT. Mice were exposed 30 min before the start of torpor, determined by the bout of the previous day. Exposure to TMT significantly (p < 0.01) blunted the fall in the minimum Tb from 28.8 ± 0.3 °C (water) to 30.1 ± 0.6 °C (TMT) and significantly (p < 0.05) decreased the amount of time Tb was under 32 °C, from 431 ± 48 min (water) to 292 ± 78 min (TMT). These results establish that mice perceived the scent of TMT as a physiologically stressful stimulus and that Tb response is modestly blunted in the presence of that stressor. Our experiment highlights the intricate interplay between predation risk and energy conservation.

Is Adenosine Action Common Ground for NREM Sleep, Torpor, and Other Hypometabolic States?

This review compares two states that lower energy expenditure: non-rapid eye movement (NREM) sleep and torpor. Knowledge on mechanisms common to these states, and particularly on the role of adenosine in NREM sleep, may ultimately open the possibility of inducing a synthetic torpor-like state in humans for medical applications and long-term space travel. To achieve this goal, it will be important, in perspective, to extend the study to other hypometabolic states, which, unlike torpor, can also be experienced by humans.

Alternate-day feeding leads to improved glucose regulation on fasting days without significant weight loss in genetically obese mice.

Alternate-day fasting (ADF) is effective for weight loss and increases insulin sensitivity in diet-induced obese rodents. However, the efficacy of ADF in genetic models of obesity has not been comprehensively studied. Mice that are deficient in leptin (ob/ob mice) are obese, diabetic, and prone to deep bouts of torpor when fasted. We tested the hypotheses that an ADF protocol in ob/ob mice would result in 1) induction of torpor on fasted days, 2) minimal body weight loss if the mice experienced torpor, and 3) no improvement in glucose control in the absence of weight loss. Female ob/ob mice and littermate controls were assigned to 1) an ad libitum regimen or 2) an ADF regimen, consisting of fasting every other day with ad libitum feeding between fasts. Over a 19-day period, littermate control mice on the ADF regimen consumed the same amount of food as littermate control mice on the ad libitum regimen, whereas the ADF ob/ob mice consumed 37% less food than ad libitum ob/ob mice. Fasting days, but not fed days, led to torpor in both genotypes. Fasting days, but not fed days, led to weight loss in both genotypes relative to ad libitum controls. Fasting days, but not fed days, produced enhanced insulin sensitivity in both genotypes and normalized circulating glucose in ob/ob mice. These data demonstrate improved glucose control on fasting days with the use of ADF in a genetic model of obesity in the face of minimal weight loss.

Neurons in ventral tegmental area tonically inhibit sympathetic outflow to brown adipose tissue: possible mediation of thermogenic signals from lateral habenula.

The lateral habenula (LHb), a nucleus involved in the response to salient, especially adverse, environmental events, is implicated in brown adipose tissue (BAT) thermogenesis caused by these events. LHb-elicited thermogenesis involves a neural pathway to the lower brain stem sympathetic control center in the medullary raphé. There are no direct connections from the LHb to the medullary raphé. LHb-mediated behavioral responses involve inhibitory control over the dopamine neurons in the ventral tegmental area (VTA), mediated via an excitatory drive from the LHb to GABAergic neurons in the tail of the VTA. We hypothesized that inhibition of the VTA is also involved in LHb-mediated BAT thermogenesis. To test this hypothesis, inhibition of neurons in the VTA with muscimol increased BAT sympathetic nerve discharge by 22.0 ± 9.2 dBµV ( n = 24, P < 0.0001) and BAT temperature by 1.2 ± 0.1°C ( P < 0.001). This response was abolished by inhibition of the medullary raphé neurons with muscimol. BAT thermogenesis initiated with focal injections of bicuculline in the LHb was reversed by subsequent blockade of GABAA receptors in the VTA with bicuculline. These results suggest that, at least in anesthetized rats, neurons in the VTA tonically inhibit BAT thermogenesis via a link, presently unknown, to the medullary raphé. Removal of this VTA-initiated inhibition is an important mechanism whereby LHb neurons activate BAT thermogenesis.

The impact of deep breathing and alternate nostril breathing on heart rate variability: a human physiology laboratory.

An increase in the beat-to-beat variability of heart rate (HRV) is a robust marker of enhanced parasympathetic activity and of a calm and relaxed state. The purpose of this laboratory activity was to introduce the concept of HRV to our students, while having them address a novel question of whether two yogic breathing techniques, namely alternate nostril breathing (ANB) and standard deep breathing (DB), impact the SD of instantaneous heart rate (SDHR), a measure of HRV. Fifty-five undergraduates enrolled in a physiology course designed for nonscience majors were tasked with analyzing HR and SDHR from electrocardiograms recorded during normal breathing, DB, and ANB. A repeated-measures ANOVA showed that HR was significantly, albeit slightly, elevated from normal (74.5 ± 13.4 beats/min; means ± SD) during DB (76.5 ± 11.2 beats/min), but not during ANB (75.7 ± 10.1 beats/min). Analysis of SDHR showed significant differences between conditions (normal: 5.5 ± 2.1, DB: 8.6 ± 3.0, ANB: 7.8 ± 2.8 beats/min). The instructors further analyzed the same data set using more robust measures of HRV (SD of sequential N-N intervals, root mean square of successive differences, and high-frequency domain of HRV) to determine whether SDHR during a 2-min epoch is a sufficient measure for HRV in the undergraduate course setting. Statistical analysis for these measures showed a near identical pattern of magnitude and significance among the groups as SDHR. Our students developed a greater appreciation for the effects of breathing patterns on HRV and HR, using the simple measure of SDHR.

The heat is on: A device that reduces cold stress-induced tachycardia in laboratory mice.

Mouse vivaria are typically maintained at an ambient temperature (Ta) of 20-26 °C which is comfortable for human researchers. However, as this Ta is well below the mouse thermoneutral zone (TNZ) of 30-32 °C, typical vivarium temperatures result in cold stress for mice. Recently, a cage has been developed that provides variable cage floor heating, allowing mice to behaviorally regulate body temperature through thermotaxis. A hand warmer provides supplemental heat, elevating cage floor surface temperature for 13 + hours up to 30 °C. This provides a heated surface for the entirety of the light phase. Here, we test the ability of these local heat sources to remove physiological signs of cold stress in mice housed at room temperature by analyzing heart rate (HR), activity, and body temperature in three experimental conditions: 23 °C, 23 °C + heated surface, or 30 °C. The location of C57Bl/6 J mice within the cage was recorded using an infrared camera. In the presence of supplemental heat at a Ta of 23 °C, mice resided atop of the area of the heated surface 85 ±â€¯3% of the 12-h light phase, as compared to 7 ±â€¯2% in the absence of supplemental heat. Further, addition of supplemental heat lowered light phase HR and activity to that seen at a Ta of 30 °C. These results indicate that provision of a local heat source is successful in reducing cold-induced tachycardia in mice housed at typical vivarium temperatures without increasing the ambient temperature of the entire laboratory and subjecting researchers to heat stress.

Changes in blood glucose as a function of body temperature in laboratory mice: implications for daily torpor.

Many small mammals, such as the laboratory mouse, utilize the hypometabolic state of torpor in response to caloric restriction. The signals that relay the lack of fuel to initiate a bout of torpor are not known. Because the mouse will only enter a torpid state when calorically challenged, it may be that one of the inputs for initiation into a bout of torpor is the lack of the primary fuel (glucose) used to power brain metabolism in the mouse. Using glucose telemetry in mice, we tested the hypotheses that 1) circulating glucose (GLC), core body temperature (Tb), and activity are significantly interrelated; and 2) that the level of GLC at the onset of torpor differs from both GLC during arousal from torpor and during feeding when there is no torpor. To test these hypotheses, six C57Bl/6J mice were implanted with glucose telemeters and exposed to different feeding conditions (ad libitum, fasting, limited food intake, and refeeding) to create different levels of GLC and Tb. We found a strong positive and linear correlation between GLC and Tb during ad libitum feeding. Furthermore, mice that were calorically restricted entered torpor bouts readily. GLC was low during torpor entry but did not drop precipitously as Tb did at the onset of a torpor bout. GLC significantly increased during arousal from torpor, indicating the presence of endogenous glucose production. While low GLC itself was not predictive of a bout of torpor, hyperactivity and low GLC preceded the onset of torpor, suggesting that this may be involved in triggering torpor.

Clocks and meals keep mice from being cool.

Daily torpor is used by small mammals to reduce daily energy expenditure in response to energetic challenges. Optimizing the timing of daily torpor allows mammals to maximize its energetic benefits and, accordingly, torpor typically occurs in the late night and early morning in most species. However, the regulatory mechanisms underlying such temporal regulation have not been elucidated. Direct control by the circadian clock and indirect control through the timing of food intake have both been suggested as possible mechanisms. Here, feeding cycles outside of the circadian range and brain-specific mutations of circadian clock genes (Vgat-Cre+ CK1δfl/fl εfl/+ ; Vgat-Cre+ Bmal1fl/fl ) were used to separate the roles of the circadian clock and food timing in controlling the timing of daily torpor in mice. These experiments revealed that the timing of daily torpor is transiently inhibited by feeding, while the circadian clock is the major determinant of the timing of torpor. Torpor never occurred during the early part of the circadian active phase, but was preferentially initiated late in the subjective night. Food intake disrupted torpor in the first 4-6 h after feeding by preventing or interrupting torpor bouts. Following interruption, re-initiation of torpor was unlikely until after the next circadian active phase. Overall, these results demonstrate that feeding transiently inhibits torpor while the central circadian clock gates the timing of daily torpor in response to energetic challenges by restricting the initiation of torpor to a specific circadian phase.

Heart rate dynamics in a marsupial hibernator.

The eastern pygmy possum (Cercartetus nanus) is a small marsupial that can express spontaneous short bouts of torpor, as well as multi-day bouts of deep hibernation. To examine heart rate (fH) control at various stages of torpor in a marsupial hibernator, and to see whether fH variability differs from that of deep placental hibernators, we used radiotelemetry to measure ECG and body temperature (Tb) while measuring the rate of O2 consumption and ventilation. fH and O2 consumption rate during euthermia were at a minimum (321±34 beats min-1, 0.705±0.048 ml O2 g-1 h-1) at an ambient temperature (Ta) of 31°C. fH had an inverse linear relationship with Ta to a maximum of 630±19 beats min-1 at a Ta of 20°C. During entry into torpor at a Ta of 20°C, fH slowed primarily as a result of episodic periods of cardiac activity where electrical activity of the heart occurred in groups of 3 or 4 heart beats. When Tb was stable at 24°C in these torpor bouts, the episodic nature of fH had disappeared (i.e. no asystoles) with a rate of 34±3 beats min-1 For multi-day bouts of deep torpor, Ta was lowered to 6.6±0.8°C. During these deep bouts of torpor, Tb reached a minimum of 8.0±1.0°C, with a minimum fH of 8 beats min-1 and a minimum O2 consumption rate of 0.029±0.07 ml O2 g-1 h-1 Shivering bouts occurred in deep torpor about every 8 min, during which ventilation occurred, and fH was elevated to 40 beats min-1 The duration of the QRS complex increased from 12 ms during euthermia to 69 ms at a Tb of 8°C. These findings demonstrate the dynamic functioning range of fH to be about 600 beats min-1 (∼80-fold), one of the largest known ranges in mammals. Our study shows that despite a separation of ∼160 million years, the control and function of the cardiac system seems indistinguishable in marsupial and placental hibernating mammals.

Group housing and nest building only slightly ameliorate the cold stress of typical housing in female C57BL/6J mice.

Huddling and nest building are two methods of behavioral thermoregulation used by mice under cold stress. In the laboratory, mice are typically housed at an ambient temperature (Ta) of 20°C, well below the lower end of their thermoneutral zone. We tested the hypothesis that the thermoregulatory benefits of huddling and nest building at a Ta of 20°C would ameliorate this cold stress compared with being singly housed at 20°C as assessed by heart rate (HR), blood pressure (BP), triiodothyronine (T3), brown adipose (BAT) expression of Elovl3 mRNA, and BAT lipid content. A series of experiments using C57BL/6J female mice exposed to 20°C in the presence or absence of nesting material and/or cage mates was used to test this hypothesis. Mice showed large differences in HR, BP, shivering, and core body temperature (Tb) when comparing singly housed mice at 20°C and 30°C, but only a modest reduction in HR with the inclusion of cage mates or bedding. However, group housing and/or nesting at 20°C decreased T3 levels compared with singly housed mice at 20°C. Singly housed mice at 20°C had a 22-fold higher level of BAT Elovl3 mRNA expression and a significantly lower triacylglycerol (TAG) content of BAT compared with singly housed mice at 30°C. Group housing at 20°C led to blunted changes in both Elovl3 mRNA and TAG levels. These findings suggest that huddling and nest building have a limited effect to ameliorate the cold stress associated with housing at 20°C.

Chronic rapamycin treatment causes diabetes in male mice.

Current evidence indicates that the mammalian target of rapamycin inhibitor rapamycin both increases longevity and, seemingly contradictorily, impairs glucose homeostasis. Most studies exploring the dimensions of this paradox have been based on rapamycin treatment in mice for up to 20 wk. We sought to better understand the metabolic effects of oral rapamycin over a substantially longer period of time in HET3 mice. We observed that treatment with rapamycin for 52 wk induced diabetes in male mice, characterized by hyperglycemia, significant urine glucose levels, and severe glucose and pyruvate intolerance. Glucose intolerance occurred in male mice by 4 wk on rapamycin and could be only partially reversed with cessation of rapamycin treatment. Female mice developed moderate glucose intolerance over 1 yr of rapamycin treatment, but not diabetes. The role of sex hormones in the differential development of diabetic symptoms in male and female mice was further explored. HET3 mice treated with rapamycin for 52 wk were gonadectomized and monitored over 10 wk. Castrated male mice remained glucose intolerant, while ovariectomized females developed significant glucose intolerance over the same time period. Subsequent replacement of 17ß-estradiol (E2) in ovariectomized females promoted a recovery of glucose tolerance over a 4-wk period, suggesting the protective role of E2 against rapamycin-induced diabetes. These results indicate that 1) oral rapamycin treatment causes diabetes in male mice, 2) the diabetes is partially reversible with cessation of treatment, and 3) E2 plays a protective role against the development of rapamycin-induced diabetes.

Activation of oxytocinergic neurons enhances torpor in mice.

Mus musculus enters a torpid state in response to caloric restriction in sub-thermoneutral ambient temperatures. This torpid state is characterized by an adaptive and controlled decrease in metabolic rate, heart rate, body temperature, and activity. Previous research has identified the paraventricular nucleus (PVN) within the hypothalamus, a region containing oxytocin neurons, as a location that is active during torpor onset. We hypothesized that oxytocin neurons within the PVN are part of this neural circuit and that activation of oxytocin neurons would deepen and lengthen torpor bouts. We report that activation of oxytocin neurons alone is not sufficient to induce a torpor-like state in the fed mouse, with no significant difference in body temperature or heart rate upon activation of oxytocin neurons. However, we found that activation of oxytocin neurons prior to the onset of daily torpor both deepens and lengthens the subsequent bout, with a 1.7 ± 0.4 °C lower body temperature and a 135 ± 32 min increase in length. We therefore conclude that oxytocin neurons are involved in the neural circuitry controlling daily torpor in the mouse.

Methods to estimate body temperature and energy expenditure dynamics in fed and fasted laboratory mice: effects of sleep deprivation and light exposure.

Monitoring body temperature and energy expenditure in freely-moving laboratory mice remains a powerful methodology used widely across a variety of disciplines-including circadian biology, sleep research, metabolic phenotyping, and the study of body temperature regulation. Some of the most pronounced changes in body temperature are observed when small heterothermic species reduce their body temperature during daily torpor. Daily torpor is an energy saving strategy characterized by dramatic reductions in body temperature employed by mice and other species when challenged to meet energetic demands. Typical measurements used to describe daily torpor are the measurement of core body temperature and energy expenditure. These approaches can have drawbacks and developing alternatives for these techniques provides options that can be beneficial both from an animal-welfare and study-complexity perspective. First, this paper presents and assesses a method to estimate core body temperature based on measurements of subcutaneous body temperature, and second, a separate approach to better estimate energy expenditure during daily torpor based on core body temperature. Third, the effects of light exposure during the habitual dark phase and sleep deprivation during the light period on body temperature dynamics were tested preliminary in fed and fasted mice. Together, the here-published approaches and datasets can be used in the future to assess body temperature and metabolism in freely-moving laboratory mice.

Induction of torpor: mimicking natural metabolic suppression for biomedical applications.

Mammalian hibernation consists of periods of depressed metabolism and reduced body temperature called "torpor" that are interspersed by normothermic arousal periods. Numerous cellular processes are halted during torpor, including transcription, translation, and ion homeostasis. Hibernators are able to survive long periods of low blood flow and body temperature followed by rewarming and reperfusion without overt signs of organ injury, which makes these animals excellent models for application of natural protective mechanisms to human medicine. This review examines efforts to induce torpor-like states in non-hibernating species using pharmacological compounds. Elucidating the underlying mechanisms of natural and pharmacologically induced torpor will speed the development of new clinical approaches to treat a variety of trauma and stress states in humans.

Central adenosine receptor signaling is necessary for daily torpor in mice.

When calorically restricted at cool ambient temperatures, mice conserve energy by entering torpor, during which metabolic rate (MR), body temperature (T(b)), heart rate (HR), and locomotor activity (LMA) decrease. Treatment with exogenous adenosine produces a similar hypometabolic state. In this study, we conducted a series of experiments using the nonspecific adenosine receptor antagonists aminophylline and 8-sulfophenyltheophylline (8-SPT) to test the hypothesis that adenosine signaling is necessary for torpor in fasted mice. In the first experiment, mice were subcutaneously infused with aminophylline while T(b), HR, and LMA were continuously monitored using implanted radiotelemeters. During a 23-h fast, saline-treated mice were torpid for 518 ± 43 min, whereas aminophylline-treated mice were torpid for significantly less time (54 ± 20 min). In a second experiment, aminophylline was infused subcutaneously into torpid mice to test the role of adenosine in the maintenance of torpor. Aminophylline reversed the hypometabolism, hypothermia, bradycardia, and hypoactivity of torpor, whereas saline did not. In the third and fourth experiments, the polar adenosine antagonist 8-SPT, which does not cross the blood-brain barrier, was infused either subcutaneously or intracerebroventricularly to test the hypothesis that both peripheral and central adenosine receptor signaling are necessary for the maintenance of torpor. Intracerebroventricular, but not subcutaneous, infusion of 8-SPT causes a return to euthermia. These findings support the hypothesis that adenosine is necessary for torpor in mice and further suggest that whereas peripheral adenosine signaling is not necessary for the maintenance of torpor, antagonism of central adenosine is sufficient to disrupt torpor.

Health Effects of Alternate Day Fasting Versus Pair-Fed Caloric Restriction in Diet-Induced Obese C57Bl/6J Male Mice.

Alternate day fasting (ADF) induces weight loss and improves various markers of health in rodents and humans. However, it is unclear whether the benefits of ADF are derived from the lower caloric intake of ADF or from the 24-h fasting period. Therefore, this study directly compared selected markers for health - such as glucose control, body weight, liver triglycerides, T cell frequencies, and others - in high-fat (60% calories from fat) diet-induced obese mice subjected to either ADF or caloric restriction (CR). Obese mice were randomly assigned to one of four groups: (1) ADF: remained on the high-fat diet, but fed on alternate days (n = 5), (2) PF: remained on the high-fat diet, but pair-fed to the ADF group (n = 5), (3) LF: moved to a chow ad libitum diet (n = 5; 17% calories from fat), and (4) HF: remained on the high-fat ad libitum diet (n = 5). An additional group of non-obese mice maintained on a chow diet since weaning were used as controls (CON: n = 5). After 10 weeks, ADF, PF, and LF mice ate fewer kcals, had a lower body mass, had smaller epididymal fat pads, improved glucose tolerance, and had a lower hepatic triglyceride content relative to HF mice (p < 0.05), but none reached that of CON mice in these measures. T cell frequencies of the spleen, blood, and mesenteric lymph nodes were reduced in ADF, PF, and HF compared to the CON group. Importantly, there were no significant differences between the ADF and PF groups in any of the measurements made in the current study. These data suggest that ADF, PF, and LF diets each lead to improved markers of health relative to high-fat diet-induced obese mice, and that the caloric restriction associated with ADF is the major factor for the noted improvements.

Resveratrol treatment in mice does not elicit the bradycardia and hypothermia associated with calorie restriction.

Dietary supplementation with resveratrol may produce calorie restriction-like effects on metabolic and longevity endpoints in mice. In this study, we sought to determine whether resveratrol treatment elicited other hallmark changes associated with calorie restriction, namely bradycardia and decreased body temperature. We found that during short-term treatment, wild-type mice on a calorie-restricted diet experienced significant decreases in both heart rate and body temperature after only 1 day whereas those receiving resveratrol exhibited no such change after 1 wk. We also used ob/ob mice to study the effects of long-term treatment because previous studies had indicated the therapeutic value of resveratrol against the linked morbidities of obesity and diabetes. After 12 wk, resveratrol treatment had produced no changes in either heart rate or body temperature. Strikingly, and in contrast to previous findings, we found that resveratrol-treated mice had significantly reduced endurance in a treadmill test. Quantitative reverse transcriptase-polymerase chain reaction suggested that a proposed target of resveratrol, Sirt1, was activated in resveratrol-treated ob/ob mice. Thus, we conclude that the bradycardia and hypothermia associated with calorie restriction occur through mechanisms unaffected by the actions of resveratrol and that further studies are needed to examine the differential effects of resveratrol in a leptin-deficient background.

Effect of stevia on the gut microbiota and glucose tolerance in a murine model of diet-induced obesity.

Artificial sweeteners have been shown to induce glucose intolerance by altering the gut microbiota; however, little is known about the effect of stevia. Here, we investigate whether stevia supplementation induces glucose intolerance by altering the gut microbiota in mice, hypothesizing that stevia would correct high fat diet-induced glucose intolerance and alter the gut microbiota. Mice were split into four treatment groups: low fat, high fat, high fat + saccharin and high fat + stevia. After 10 weeks of treatment, mice consuming a high fat diet (60% kcal from fat) developed glucose intolerance and gained more weight than mice consuming a low fat diet. Stevia supplementation did not impact body weight or glucose intolerance. Differences in species richness and relative abundances of several phyla were observed in low fat groups compared to high fat, stevia and saccharin. We identified two operational taxonomic groups that contributed to differences in beta-diversity between the stevia and saccharin groups: Lactococcus and Akkermansia in females and Lactococcus in males. Our results demonstrate that stevia does not rescue high fat diet-induced changes in glucose tolerance or the microbiota, and that stevia results in similar alterations to the gut microbiota as saccharin when administered in concordance with a high fat diet.