Gut-derived glucagon like peptide-1 (GLP-1) acts in the postprandial period to stimulate insulin secretion and inhibit gastrointestinal motor and secretory function; whether endogenous peripheral GLP-1 inhibits food intake is less clear. We hypothesized that GLP-1 inhibits food intake in the fed, but not fasted, state. There is evidence that GLP-1 acts via stimulation of vagal afferent neurons (VAN); we further hypothesized that the satiating effects of endogenous GLP-1 in the postprandial period is determined either by a change in GLP-1 receptor (GLP-1R) expression or localization to different cellular compartments in VAN.

The vagal afferent pathway senses hormones released from the gut in response to nutritional cues and relays these signals to the brain. We tested the hypothesis that leptin resistance in vagal afferent neurons (VAN) is responsible for the onset of hyperphagia by developing a novel conditional knockout mouse to delete leptin receptor selectively in sensory neurons (Nav1.8/LepR (fl/fl) mice). Chow fed Nav1.8/LepR (fl/fl) mice weighed significantly more and had increased adiposity compared with wildtype mice. Cumulative food intake, meal size, and meal duration in the dark phase were increased in Nav1.8/LepR (fl/fl) mice; energy expenditure was unaltered. Reduced satiation in Nav1.8/LepR (fl/fl) mice is in part due to reduced sensitivity of VAN to CCK and the subsequent loss of VAN plasticity. Crucially Nav1.8/LepR (l/fl) mice did not gain further weight in response to a high fat diet. We conclude that disruption of leptin signaling in VAN is sufficient and necessary to promote hyperphagia and obesity.

ShcKO mice have low body fat and resist weight gain on a high fat diet, indicating that Shc proteins may influence enzymes involved in β-oxidation. To investigate this idea, the activities of β-oxidation and ketone body metabolism enzymes were measured.

While it has been proposed that Shc family of adaptor proteins may influence aging by regulating insulin signaling and energy metabolism, the overall impact of Shc proteins on whole body energy metabolism has yet to be elucidated. Thus, the purpose of this study was to determine the influence of Shc proteins and aging on whole body energy metabolism in a mouse model under ambient conditions (22°C) and acute cold exposure (12°C for 24 hours). Using indirect respiration calorimetry, we investigated the impact of Shc proteins and aging on EE and substrate utilization (RQ) in p66 Shc-/- (ShcKO) and wild-type (WT) mice. Calorimetry measurements were completed in 3, 15, and 27 mo mice at 22°C and 12°C. At both temperatures and when analyzed across all age groups, ShcKO mice demonstrated lower 24 h total EE values than that of WT mice when EE data was expressed as either kJ per mouse, or adjusted by body weight or crude organ mass (ORGAN) (P≤0.01 for all). The ShcKO mice also had higher (P<0.05) fed state RQ values than WT animals at 22°C, consistent with an increase in glucose utilization. However, Shc proteins did not influence age-related changes in energy expenditure or RQ. Age had a significant impact on EE at 22°C, regardless of how EE data was expressed (P<0.05), demonstrating a pattern of increase in EE from age 3 to 15 mo, followed by a decrease in EE at 27 mo. These results indicate a decline in whole body EE with advanced age in mice, independent of changes in body weight (BW) or fat free mass (FFM). The results of this study indicate that both Shc proteins and aging should be considered as factors that influence energy expenditure in mice.

The Amargosa vole is a highly endangered rodent endemic to a small stretch of the Amargosa River basin in Inyo County, California. It specializes on a single, nutritionally marginal food source in nature. As part of a conservation effort to preserve the species, a captive breeding population was established to serve as an insurance colony and a source of individuals to release into the wild as restored habitat becomes available. The colony has successfully been maintained on commercial diets for multiple generations, but there are concerns that colony animals could lose gut microbes necessary to digest a wild diet. We analyzed feces from colony-reared and recently captured wild-born voles on various diets, and foregut contents from colony and wild voles. Unexpectedly, fecal microbial composition did not greatly differ despite drastically different diets and differences observed were mostly in low-abundance microbes. In contrast, colony vole foregut microbiomes were dominated by Allobaculum sp. while wild foreguts were dominated by Lactobacillus sp. If these bacterial community differences result in beneficial functional differences in digestion, then captive-reared Amargosa voles should be prepared prior to release into the wild to minimize or eliminate those differences to maximize their chance of success.

Alterations in markers of mitochondrial content with ketogenic diets (KD) have been reported in tissues of rodents, but morphological quantification of mitochondrial mass using transmission electron microscopy (TEM), the gold standard for mitochondrial quantification, is needed to further validate these findings and look at specific regions of interest within a tissue. In this study, red gastrocnemius muscle, the prefrontal cortex, the hippocampus, and the liver left lobe were used to investigate the impact of a 1-month KD on mitochondrial content in healthy middle-aged mice. The results showed that in red gastrocnemius muscle, the fractional area of both subsarcolemmal (SSM) and intermyofibrillar (IMM) mitochondria was increased, and this was driven by an increase in the number of mitochondria. Mitochondrial fractional area or number was not altered in the liver, prefrontal cortex, or hippocampus following 1 month of a KD. These results demonstrate tissue-specific changes in mitochondrial mass with a short-term KD and highlight the need to study different muscle groups or tissue regions with TEM to thoroughly determine the effects of a KD on mitochondrial mass.

Women represent the majority of Alzheimer's disease patients and show typical symptoms. Genetic, hormonal, and behavioral mechanisms have been proposed to explain sex differences in dementia prevalence. However, whether sex differences exist in the epigenetic landscape of neuronal tissue during the progression of the disease is still unknown.

There is increasing evidence that Shc proteins play a role in energy metabolism, and we have previously reported that knockdown of Shc proteins influences the energetic response to acute (3 days) calorie restriction (CR) in 18-month-old mice. Whether Shc proteins play a role in the metabolic response to CR in younger mice has yet to be elucidated. Hence, we sought to determine the impact of 3 days and longer term (2 months) CR on energy expenditure (EE) and respiratory quotient (RQ) in 3 month-old Shc knockout (ShcKO) and wild-type (WT) mice. ShcKO mice decreased (P < 0.001) EE normalized for body weight (EEBW) by 3 days of CR, while no such change was observed in WT animals. However, both ShcKO and WT mice decreased (P < 0.001) EEBW at 2 months of CR and there were no differences in body weight between the ShcKO and WT mice at either 3 days or 2 months of CR. Consistent with increased fatty acid oxidation, only ShcKO mice maintained decreased (P < 0.001) 24 h RQ through 2 months of CR, suggesting that they were able to maintain increased fatty acid oxidation for a longer period of time than WT mice. These results indicate that Shc proteins may contribute to some of the acute energetic responses to CR.

Calorie restriction, without malnutrition, has been shown to increase lifespan and is associated with a shift away from glycolysis toward beta-oxidation. The objective of this study was to mimic this metabolic shift using low-carbohydrate diets and to determine the influence of these diets on longevity and healthspan in mice. C57BL/6 mice were assigned to a ketogenic, low-carbohydrate, or control diet at 12 months of age and were either allowed to live their natural lifespan or tested for physiological function after 1 or 14 months of dietary intervention. The ketogenic diet (KD) significantly increased median lifespan and survival compared to controls. In aged mice, only those consuming a KD displayed preservation of physiological function. The KD increased protein acetylation levels and regulated mTORC1 signaling in a tissue-dependent manner. This study demonstrates that a KD extends longevity and healthspan in mice.

Mutations in the X-linked gene MECP2 cause the majority of Rett syndrome (RTT) cases. Two differentially spliced isoforms of exons 1 and 2 (MeCP2-e1 and MeCP2-e2) contribute to the diverse functions of MeCP2, but only mutations in exon 1, not exon 2, are observed in RTT. We previously described an isoform-specific MeCP2-e1-deficient male mouse model of a human RTT mutation that lacks MeCP2-e1 while preserving expression of MeCP2-e2. However, RTT patients are heterozygous females that exhibit delayed and progressive symptom onset beginning in late infancy, including neurologic as well as metabolic, immune, respiratory and gastrointestinal phenotypes. Consequently, we conducted a longitudinal assessment of symptom development in MeCP2-e1 mutant females and males. A delayed and progressive onset of motor impairments was observed in both female and male MeCP2-e1 mutant mice, including hind limb clasping and motor deficits in gait and balance. Because these motor impairments were significantly impacted by age-dependent increases in body weight, we also investigated metabolic phenotypes at an early stage of disease progression. Both male and female MeCP2-e1 mutants exhibited significantly increased body fat compared to sex-matched wild-type littermates prior to weight differences. Mecp2e1-/y males exhibited significant metabolic phenotypes of hypoactivity, decreased energy expenditure, increased respiratory exchange ratio, but decreased food intake compared to wild-type. Untargeted analysis of lipid metabolites demonstrated a distinguishable profile in MeCP2-e1 female mutant liver characterized by increased triglycerides. Together, these results demonstrate that MeCP2-e1 mutation in mice of both sexes recapitulates early and progressive metabolic and motor phenotypes of human RTT.

We studied ubiquinone (Q), Q homologue ratio, and steady-state levels of mCOQ transcripts in tissues from mice fed ad libitum or under calorie restriction. Maximum ubiquinone levels on a protein basis were found in kidney and heart, followed by liver, brain, and skeletal muscle. Liver and skeletal muscle showed the highest Q(9)/Q(10) ratios with significant interindividual variability. Heart, kidney, and particularly brain exhibited lower Q(9)/Q(10) ratios and interindividual variability. In skeletal muscle and heart, the most abundant mCOQ transcript was mCOQ7, followed by mCOQ8, mCOQ2, mPDSS2, mPDSS1, and mCOQ3. In nonmuscular tissues (liver, kidney, and brain) the most abundant mCOQ transcript was mCOQ2, followed by mCOQ7, mCOQ8, mPDSS1, mPDSS2, and mCOQ3. Calorie restriction increased both ubiquinone homologues and mPDSS2 mRNA in skeletal muscle, but mCOQ7 was decreased. In contrast, Q(9) and most mCOQ transcripts were decreased in heart. Calorie restriction also modified the Q(9)/Q(10) ratio, which was increased in kidney and decreased in heart without alterations in mPDSS1 or mPDSS2 transcripts. We demonstrate for the first time that unique patterns of mCOQ transcripts exist in muscular and nonmuscular tissues and that Q and COQ genes are targets of calorie restriction in a tissue-specific way.

It has been proposed that Shc proteins may influence aging by regulating insulin signaling and energy metabolism. Evidence suggests that deletion of p66Shc could partially attenuate weight gain on a high fat diet by increasing energy expenditure. However, the impact of p66Shc on the metabolic response to calorie restriction (CR) has not been determined. Thus, we used indirect respiration calorimetry to determine the impact of CR on energy expenditure (EE) and substrate utilization (RQ) in 18mo p66Shc(-/-) and wild-type (WT) mice. Calorimetry measurements were completed at baseline and following 3d of 40% CR and 2 mo of 26% CR. There was no difference (P>0.10) in EE and RQ between gentoypes, regardless of how EE data was normalized. Both p66Shc(-/-) and WT mice showed decreases (P<0.001) in EE normalized for body weight at 2 mo of CR. However, the response to 3d of CR was different between genotypes with only the p66Shc(-/-) showing a decrease (P<0.001) in 24 h EE expressed per mouse or normalized for body weight. The results indicate that p66Shc does not significantly influence EE in 18 mo mice at baseline or 2 mo of CR, although it may play a role in the EE response to very acute CR.

Consumption of high-fat diet (HF) leads to hyperphagia and increased body weight in male rodents. Female rodents are relatively resistant to hyperphagia and weight gain in response to HF, in part via effects of estrogen that suppresses food intake and increases energy expenditure. However, sex differences in energy expenditure and activity levels with HF challenge have not been systemically described. We hypothesized that, in response to short-term HF feeding, female mice will have a higher energy expenditure and be more resistant to HF-induced hyperphagia than male mice.

The causes of the decline in skeletal muscle mass and function with age, known as sarcopenia, are poorly understood. Nutrition (calorie restriction) interventions impact many cellular processes and increase lifespan and preserve muscle mass and function with age. As we previously observed an increase in life span and muscle function in aging mice on a ketogenic diet (KD), we aimed to investigate the effect of a KD on the maintenance of skeletal muscle mass with age and the potential molecular mechanisms of this action. Twelve-month-old mice were assigned to an isocaloric control or KD until 16 or 26 months of age, at which time skeletal muscle was collected for evaluating mass, morphology, and biochemical properties. Skeletal muscle mass was significantly greater at 26 months in the gastrocnemius of mice on the KD. This result in KD mice was associated with a shift in fiber type from type IIb to IIa fibers and a range of molecular parameters including increased markers of NMJ remodeling, mitochondrial biogenesis, oxidative metabolism, and antioxidant capacity, while decreasing endoplasmic reticulum (ER) stress, protein synthesis, and proteasome activity. Overall, this study shows the effectiveness of a long-term KD in mitigating sarcopenia. The diet preferentially preserved oxidative muscle fibers and improved mitochondrial and antioxidant capacity. These adaptations may result in a healthier cellular environment, decreasing oxidative and ER stress resulting in less protein turnover. These shifts allow mice to better maintain muscle mass and function with age.

The effect of a ketogenic diet (KD) on middle aged female mice is poorly understood as most of this work have been conducted in young female mice or diseased models. We have previously shown that an isocaloric KD started at middle age in male mice results in enhanced mitochondrial mass and function after 2 months on diet and improved cognitive behavior after being on diet for 14 months when compared with their control diet (CD) fed counterparts. Here, we aimed to investigate the effect of an isocaloric 2-month KD or CD on healthy 14-month-old female mice. At 16 months of age cognitive behavior tests were performed and then serum, skeletal muscle, cortex, and hippocampal tissues were collected for biochemical analysis. Two months on a KD resulted in enhanced cognitive behavior associated with anxiety, memory, and willingness to explore. The improved neurocognitive function was associated with increased PGC1α protein in the gastrocnemius (GTN) muscle and nuclear fraction. The KD resulted in a tissue specific increase in mitochondrial mass and kynurenine aminotransferase (KAT) levels in the GTN and soleus muscles, with a corresponding decrease in kynurenine and increase in kynurenic acid levels in serum. With KAT proteins being responsible for converting kynurenine into kynurenic acid, which is unable to cross the blood brain barrier and be turned into quinolinic acid-a potent neurotoxin, this study provides a potential mechanism of crosstalk between muscle and brain in mice on a KD that may contribute to improved cognitive function in middle-aged female mice.

Declines in mitochondrial mass are thought to be a hallmark of mammalian aging, and a ketogenic diet (KD) may prevent the age-related decreases in mitochondrial content. The objective of this study was to investigate the impact of a KD on markers of mitochondrial mass. Mice were fed an isocaloric control diet (CD) or KD from 12 months of age. Tissues were collected after 1 month and 14 months of intervention, and a panel of commonly used markers of mitochondrial mass (mitochondrial enzyme activities and levels, mitochondrial to nuclear DNA ratio, and cardiolipin content) were measured. Our results showed that a KD stimulated activities of marker mitochondrial enzymes including citrate synthase, Complex I, and Complex IV in hindlimb muscle in aged mice. KD also increased the activity of citrate synthase and prevented an age-related decrease in Complex IV activity in aged brain. No other markers were increased in these tissues. Furthermore, the impacts of a KD on liver and kidney were mixed with no pattern indicative of a change in mitochondrial mass. In conclusion, results of the present study suggest that a KD induces tissue-specific changes in mitochondrial enzyme activities, or structure, rather than global changes in mitochondrial mass across tissues.

Studies have shown ketogenic diets (KD) started from early middle-age improved health span and longevity in mice. KDs started later in life or administered intermittently may be more feasible and promote compliance. Therefore, this study sought to test if continuous or intermittent KDs started in late-middle-aged mice would improve cognition and motor function at advanced age. Eighteen-month-old male C57BL/6JN mice were assigned to an isocaloric control (CD), KD, or intermittent ketogenic (IKD, 3-day KD/week) diet. A panel of behavior tests were performed to assess cognitive and motor functions with aging. Y-maze alternation rate was higher for both IKD and KD mice at 23 months of age and for KD mice at 26 months indicating an improved spatial working memory. Twenty-six-month-old KD mice also showed better spatial learning memory in Barnes maze when compared to the CD. Improved grid wire hang performance was observed in aged IKD and KD versus CD mice indicating better muscle endurance under isometric contraction. A reduced level of circulating proinflammatory cytokines in aged KD (IL-6 and TNF-α) and IKD (IL-6) mice may contribute to the phenotypic improvements observed with these interventions. This study demonstrates that when initiated at late-middle age, the KD improved measures of spatial memory and grid wire hang performance in aged male mice, with IKD showing results intermediate to the CD and KD groups.

An epidemic of obesity has affected large portions of the world, increasing the risk of developing many different age-associated diseases, including cancer, cardiovascular disease, and diabetes. In contrast with the prevailing notion that "a calorie is just a calorie," there are clear differences, within and between individuals, in the metabolic response to different macronutrient sources. Recent findings challenge this oversimplification; calories from different macronutrient sources or consumed at different times of day have metabolic effects beyond their value as fuel. Here, we summarize discussions conducted at a recent NIH workshop that brought together experts in calorie restriction, macronutrient composition, and time-restricted feeding to discuss how dietary composition and feeding schedule impact whole-body metabolism, longevity, and healthspan. These discussions may provide insights into the long-sought molecular mechanisms engaged by calorie restriction to extend lifespan, lead to novel therapies, and potentially inform the development of a personalized food-as-medicine approach to healthy aging.

Calorie restriction (CR) consistently extends longevity and delays age-related diseases across several animal models. We have previously shown that different dietary fat sources can modulate life span and mitochondrial ultrastructure, function and membrane fatty acid composition in mice maintained on a 40% CR. In particular, animals consuming lard as the main fat source (CR-Lard) lived longer than CR mice consuming diets with soybean oil (CR-Soy) or fish oil (CR-Fish) as the predominant lipid source. In the present work, a transcriptomic analysis in the liver and skeletal muscle was performed in order to elucidate possible mechanisms underlying the changes in energy metabolism and longevity induced by dietary fat in CR mice. After 8 months of CR, transcription downstream of several mediators of inflammation was inhibited in liver. In contrast, proinflammatory signaling was increased in the CR-Fish versus other CR groups. Dietary fish oil induced a gene expression pattern consistent with increased transcriptional regulation by several cytokines (TNF, GM-CSF, TGF-β) and sex hormones when compared to the other CR groups. The CR-Fish also had lower expression of genes involved in fatty acid biosynthesis and increased expression of mitochondrial and peroxisomal fatty acid β-oxidation genes than the other CR diet groups. Our data suggest that a diet high in n-3 PUFA, partially reverts CR-related changes in gene expression of key processes, such as inflammation and steroid hormone signaling, and this may mitigate life span extension with CR in mice consuming diets high in fish oil.

Microbial dysbiosis and increased intestinal permeability are targets for prevention or reversal of weight gain in high-fat (HF) diet-induced obesity (DIO). Prebiotic milk oligosaccharides (MO) have been shown to benefit the host intestine but have not been used in DIO. We hypothesized that supplementation with bovine MO would prevent the deleterious effect of HF diet on the gut microbiota and intestinal permeability and attenuate development of the obese phenotype. C57BL/6 mice were fed a control diet, HF (40% fat/kcal), or HF + prebiotic [6%/kg bovine milk oligosaccharides (BMO) or inulin] for 1, 3, or 6 wk. Gut microbiota and intestinal permeability were assessed in the ileum, cecum, and colon. Addition of BMO to the HF diet significantly attenuated weight gain, decreased adiposity, and decreased caloric intake; inulin supplementation also lowered weight gain and adiposity, but this did not reach significance. BMO and inulin completely abolished the HF diet-induced increase in paracellular and transcellular permeability in the small and large intestine. Both BMO and inulin increased abundance of beneficial microbes Bifidobacterium and Lactobacillus in the ileum. However, inulin supplementation altered phylogenetic diversity and decreased species richness. We conclude that addition of BMO to the HF diet completely prevented increases in intestinal permeability and microbial dysbiosis and was partially effective to prevent weight gain in DIO.NEW & NOTEWORTHY This study provides the first report of the effects of prebiotic bovine milk oligosaccharides on the host phenotype of high-fat diet-induced obesity in mice.