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Studies suggest that ketogenic diets (KD) may improve memory in mouse models of aging and Alzheimer's disease (AD). This study determined whether a continuous or intermittent KD (IKD) enhanced cognitive behavior in the TgF344-AD rat model of AD. At 6 months-old, TgF344-AD and wild-type (WT) littermates were placed on a control (CD), KD, or IKD (morning CD and afternoon KD) provided as two meals per day for 2 or 6 months. Cognitive and motor behavior and circulating β-hydroxybutyrate (BHB), AD biomarkers and blood lipids were assessed. Animals on a KD diet had elevated circulating BHB, with IKD levels intermediate to CD and KD. TgF344-AD rats displayed impaired spatial learning memory in the Barnes maze at 8 and 12 months of age and impaired motor coordination at 12 months of age. Neither KD nor IKD improved performance compared to CD. At 12 months of age, TgF344-AD animals had elevated blood lipids. IKD reduced lipids to WT levels with KD further reducing cholesterol below WT levels. This study shows that at 8 or 12 months of age, KD or IKD intervention did not improve measures of cognitive or motor behavior in TgF344-AD rats; however, both IKD and KD positively impacted circulating lipids.

The Ketogenic Diet (KD) improves memory and longevity in aged C57BL/6 mice. We tested 7 months KD vs. control diet (CD) in the mouse Alzheimer's Disease (AD) model APP/PS1. KD significantly rescued Long-Term-Potentiation (LTP) to wild-type levels, not by changing Amyloid-β (Aβ) levels. KD's 'main actor' is thought to be Beta-Hydroxy-butyrate (BHB) whose levels rose significantly in KD vs. CD mice, and BHB itself significantly rescued LTP in APP/PS1 hippocampi. KD's 6 most significant pathways induced in brains by RNAseq all related to Synaptic Plasticity. KD induced significant increases in synaptic plasticity enzymes p-ERK and p-CREB in both sexes, and of brain-derived neurotrophic factor (BDNF) in APP/PS1 females. We suggest KD rescues LTP through BHB's enhancement of synaptic plasticity. LTP falls in Mild-Cognitive Impairment (MCI) of human AD. KD and BHB, because they are an approved diet and supplement respectively, may be most therapeutically and translationally relevant to the MCI phase of Alzheimer's Disease.

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.

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.

Pancreatic ductal adenocarcinoma (PDAC) continues to be a major health problem. A ketogenic diet (KD), characterized by a very low carbohydrate and high fat composition, has gained attention for its anti-tumor potential. We evaluated the effect and mechanisms of feeding a strict KD alone or in combination with gemcitabine in the autochthonous LSL-KrasG12D/+; LSL-Trp53 R172H/+; Pdx1-Cre (KPC) mouse model. For this purpose, both male and female pancreatic tumor-bearing KPC mice were allocated to a control diet (CD; %kcal: 70% carb, 14% protein, 16% fat), a KD (%kcal: 14% protein, 1% carb, 85% fat), a CD + gemcitabine (CG), or a KD + gemcitabine (KG) group. Mice fed a KD alone or in combination with gemcitabine showed significantly increased blood β-hydroxybutyrate levels compared to mice fed a CD or CG. KPC mice fed a KG had a significant increase in overall median survival compared to KPC mice fed a CD (increased overall median survival by 42%). Interestingly, when the data was disaggregated by sex, the effect of a KG was significant in female KPC mice (60% increase in median overall survival), but not in male KPC mice (28% increase in median overall survival). Mechanistically, the enhanced survival response to a KD combined with gemcitabine was multifactorial, including inhibition of ERK and AKT pathways, regulation of fatty acid metabolism and the modulation of the gut microbiota. In summary, a KD in combination with gemcitabine appears beneficial as a treatment strategy in PDAC in KPC mice, deserving further clinical evaluation.

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.

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.

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.

BackgroundWomen 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.MethodsTo investigate the differences of histone H3 modifications involved in transcription, we determined the genome-wide profiles of H3K4me3, H3K27ac, and H3K27me3 in brain cortexes of an Alzheimer mouse model (PSAPP). Gastrocnemius muscles were also tested since they are known to be different in the two sexes and are affected during the disease progression.ResultsCorrelation analysis distinguished the samples based on sex for H3K4me3 and H3K27me3 but not for H3K27ac. The analysis of transcription starting sites (TSS) signal distribution, and analysis of bounding sites revealed that gastrocnemius is more influenced than brain by sex for the three histone modifications considered, exception made for H3K27me3 distribution on the X chromosome which showed sex-related differences in promoters belonging to behavior and cellular or neuronal spheres in mice cortexes.ConclusionsH3K4me3, H3K27ac, and H3K27me3 signals are slightly affected by sex in brain, with the exception of H3K27me3, while a higher number of differences can be found in gastrocnemius.

BackgroundConsumption 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.MethodsSix-week-old male and female C57BL/6 J mice were fed either low fat (LF, 10% fat) or moderate HF (45% fat) for 5 weeks, and energy expenditure, activity and meal pattern measured using comprehensive laboratory animal monitoring system (CLAMS).ResultsAfter 5 weeks, HF-fed male mice had a significant increase in body weight and fat mass, compared with LF-fed male mice. HF-fed female had a significant increase in body weight compared with LF-fed female mice, but there was no significant difference in fat mass. HF-fed male mice had lower energy expenditure compared to HF-fed female mice, likely due in part to reduced physical activity in the light phase. HF-fed male mice also had increased energy intake in the dark phase compared to LF-fed male mice and a reduced response to exogenous cholecystokinin-induced inhibition of food intake. In contrast, there was no difference in energy intake between LF-fed and HF-fed female mice.ConclusionsThe data show that female mice are generally protected from short-term HF-induced alterations in energy balance, possibly by maintaining higher energy expenditure and an absence of hyperphagia. However, HF-feeding in male mice induced weight and fat mass gain and hyperphagia. These findings suggest that there is a sex difference in the response to short-term HF-feeding in terms of both energy expenditure and control of food intake.

Malignant hyperthermia (MH) is characterized by induction of skeletal muscle hyperthermia in response to a dysregulated increase in myoplasmic calcium. Although altered energetics play a central role in MH, MH-susceptible humans and mouse models are often described as having no phenotype until exposure to a triggering agent. The purpose of this study was to determine the influence of the R163C ryanodine receptor 1 mutation, a common MH mutation in humans, on energy expenditure, and voluntary wheel running in mice. Energy expenditure was measured by indirect respiration calorimetry in wild-type (WT) and heterozygous R163C (HET) mice over a range of ambient temperatures. Energy expenditure adjusted for body weight or lean mass was increased (P

Maintaining a healthy body weight requires an exquisite balance between energy intake and energy expenditure. To understand the genetic and environmental factors that contribute to the regulation of body weight, an important first step is to establish the normal range of metabolic values and primary sources contributing to variability. Energy metabolism is measured by powerful and sensitive indirect calorimetry devices. Analysis of nearly 10,000 wild-type mice from two large-scale experiments revealed that the largest variation in energy expenditure is due to body composition, ambient temperature, and institutional site of experimentation. We also analyze variation in 2329 knockout strains and establish a reference for the magnitude of metabolic changes. Based on these findings, we provide suggestions for how best to design and conduct energy balance experiments in rodents. These recommendations will move us closer to the goal of a centralized physiological repository to foster transparency, rigor and reproducibility in metabolic physiology experimentation.

Loss of skeletal muscle mass and function is a hallmark of aging. This phenomenon has been related to a dysregulation of mitochondrial function and proteostasis. Calorie restriction (CR) has been demonstrated to delay aging and preserve function until late in life, particularly in muscle. Recently, we reported the type of dietary fat plays an important role in determining life span extension with 40% CR in male mice. In these conditions, lard fed mice showed an increased longevity compared to mice fed soybean or fish oils. In this article, we analyze the effect of 40% CR on muscle mitochondrial mass, autophagy, and mitochondrial dynamics markers in mice fed these diets. In CR fed animals, lard preserved muscle fibers structure, mitochondrial ultrastructure, and fission/fusion dynamics and autophagy, not only compared to control animals, but also compared with CR mice fed soybean and fish oils as dietary fat. We focus our discussion on dietary fatty acid saturation degree as an essential predictor of life span extension in CR 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.

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.

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert NADH and FADH into ATP. Since mitochondria are also the primary site for oxygen use and nutrient oxidation to carbon dioxide and water, how efficiently they use oxygen and produce ATP directly relates to the efficiency of nutrient metabolism, nutrient requirements of the animal, and health of the animal. The purpose of this method is to examine mitochondrial respiration, which can be used to examine the effects of different drugs, diets and environmental effects on mitochondrial metabolism. Results include oxygen consumption measured as proton dependent respiration (State 3) and proton leak dependent respiration (State 4). The ratio of State 3 / State 4 respiration is defined as respiratory control ratio (RCR) and can represent mitochondrial energetic efficiency. Mitochondrial proton leak is a process that allows dissipation of mitochondrial membrane potential (MMP) by uncoupling oxidative phosphorylation from ADP decreasing the efficiency of ATP synthesis. Oxygen and TRMP+ sensitive electrodes with mitochondrial substrates and electron transport chain inhibitors are used to measure State 3 and State 4 respiration, mitochondrial membrane PMF (or the potential to produce ATP) and proton leak. Limitations to this method are that liver tissue must be as fresh as possible and all biopsies and assays must be performed in less than 10 h. This limits the number of samples that can be collected and processed by a single person in a day to approximately 5. However, only 1 g of liver tissue is needed, so in large animals, such as dairy cattle, the amount of sample needed is small relative to liver size and there is little recovery time needed.

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.

Shc proteins play a role in energy metabolism through interaction with the insulin receptor. The aim of this study was to determine whether Shc proteins influence liver glycolysis and gluconeogenesis under both fed and fasted states. Decreased glycolytic and increased gluconeogenic and transamination enzyme activities were observed in ShcKO versus WT mice. Levels of key regulatory metabolites, such as fructose-2,6-bisphosphate, matched the activity of metabolic pathways. Protein levels of glycolytic and gluconeogenic enzymes were not different. pAMPK protein levels increased with fasting and were higher in ShcKO versus WT mice. Therefore, Shc proteins play a role in shifting the metabolism from glucose oxidation to gluconeogenesis and lipid catabolism and should be considered as regulators of fuel selection. Fuel selection and utilization could play a critical role in healthy aging. Characterization of metabolic events in ShcKO mice would help to elucidate how metabolism is influenced by these proteins.

Calorie restriction (CR) has been repeatedly shown to prevent cancer, diabetes, hypertension, and other age-related diseases in a wide range of animals, including non-human primates and humans. In rodents, CR also increases lifespan and is a powerful tool for studying the aging process. Recently, it has been reported in mice that dietary fat plays an important role in determining lifespan extension with 40% CR. In these conditions, animals fed lard as dietary fat showed an increased longevity compared with mice fed soybean or fish oils. In this paper, we study the effect of these dietary fats on structural and physiological parameters of kidney from mice maintained on 40% CR for 6 and 18 months. Analyses were performed using quantitative electron microcopy techniques and protein expression in Western blots. CR mitigated most of the analyzed age-related parameters in kidney, such as glomerular basement membrane thickness, mitochondrial mass in convoluted proximal tubules and autophagic markers in renal homogenates. The lard group showed improved preservation of several renal structures with aging when compared to the other CR diet groups. These results indicate that dietary fat modulates renal structure and function in CR mice and plays an essential role in the determination of health span in rodents.

Calorie restriction (CR) without malnutrition extends life span in several animal models. It has been proposed that a decrease in the amount of polyunsaturated fatty acids (PUFAs), and especially n-3 fatty acids, in membrane phospholipids may contribute to life span extension with CR. Phospholipid PUFAs are sensitive to dietary fatty acid composition, and thus, the purpose of this study was to determine the influence of dietary lipids on life span in CR mice. C57BL/6J mice were assigned to four groups (a 5% CR control group and three 40% CR groups) and fed diets with soybean oil (high in n-6 PUFAs), fish oil (high in n-3 PUFAs), or lard (high in saturated and monounsaturated fatty acids) as the primary lipid source. Life span was increased (p < .05) in all CR groups compared to the Control mice. Life span was also increased (p < .05) in the CR lard mice compared to animals consuming either the CR fish or soybean oil diets. These results indicate that dietary lipid composition can influence life span in mice on CR, and suggest that a diet containing a low proportion of PUFAs and high proportion of monounsaturated and saturated fats may maximize life span in animals maintained on CR.

Shc proteins interact with the insulin receptor, indicating a role in regulating glycolysis. To investigate this idea, the activities of key glycolytic regulatory enzymes and metabolites levels were measured in skeletal muscle from mice with low levels of Shc proteins (ShcKO) and wild-type (WT) controls. The activities of hexokinase, phosphofructokinase-1 and pyruvate kinase were decreased in ShcKO versus WT mice under both fed and fasted conditions. Increased alanine transaminase and branched-chain amino acid transaminase activities were also observed in ShcKO mice under both fed and fasting conditions. Protein expression of glycolytic enzymes was unchanged in the ShcKO and WT mice, indicating that decreased activities were not due to changes in their transcription. Changes in metabolite levels were consistent with the observed changes in enzyme activities. In particular, the levels of fructose-2,6-bisphosphate, a potent activator of phosphofructokinase-1, were consistently decreased in the ShcKO mice. Furthermore, the levels of lactate (inhibitor of hexokinase and phosphofructokinase-1) and citrate (inhibitor of phosphofructokinase-1 and pyruvate kinase) were increased in fed and fasted ShcKO versus WT mice. Pyruvate dehydrogenase activity was lower in ShcKO versus WT mice under fed conditions, and showed inhibition under fasting conditions in both ShcKO and WT mice, with ShcKO mice showing less inhibition than the WT mice. Pyruvate dehydrogenase kinase 4 levels were unchanged under fed conditions but were lower in the ShcKO mice under fasting conditions. These studies indicate that decreased levels of Shc proteins in skeletal muscle lead to a decreased glycolytic capacity in both fed and fasted states.

The signaling molecule p66Shc is often described as a longevity protein. This conclusion is based on a single life span study that used a small number of mice. The purpose of the present studies was to measure life span in a sufficient number of mice to determine if longevity is altered in mice with decreased Shc levels (ShcKO). Studies were completed at UC Davis and the European Institute of Oncology (EIO). At UC Davis, male C57BL/6J WT and ShcKO mice were fed 5% or 40% calorie-restricted (CR) diets. In the 5% CR group, there was no difference in survival curves between genotypes. There was also no difference between genotypes in prevalence of neoplasms or other measures of end-of-life pathology. At 40% calorie restriction group, 70th percentile survival was increased in ShcKO, while there were no differences between genotypes in median or subsequent life span measures. At EIO, there was no increase in life span in ShcKO male or female mice on C57BL/6J, 129Sv, or hybrid C57BL/6J-129Sv backgrounds. These studies indicate that p66Shc is not a longevity protein. However, additional studies are needed to determine the extent to which Shc proteins may influence the onset and severity of specific age-related diseases.

In this paper we analyzed changes in hepatocyte mitochondrial mass and ultrastructure as well as in mitochondrial markers of fission/fusion and biogenesis in mice subjected to 40% calorie restriction (CR) for 18 months versus ad libitum-fed controls. Animals subjected to CR were separated into three groups with different dietary fats: soybean oil (also in controls), fish oil and lard. Therefore, the effect of the dietary fat under CR was studied as well. Our results show that CR induced changes in hepatocyte and mitochondrial size, in the volume fraction occupied by mitochondria, and in the number of mitochondria per hepatocyte. Also, mean number of mitochondrial cristae and lengths were significantly higher in all CR groups compared with controls. Finally, CR had no remarkable effects on the expression levels of fission and fusion protein markers. However, considerable differences in many of these parameters were found when comparing the CR groups, supporting the idea that dietary fat plays a relevant role in the modulation of CR effects in aged mice.

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.

The predisposition of cats to gain weight following neutering is well established; however, there is little information about the distribution and range of post-neutering weight gains observed in cats under a controlled environment. This retrospective study investigated 6-month post-castration weight gain and distribution of percentage body weight (BW) change in a cohort of twenty, male domestic shorthair cats relative to a control group of intact cats. Cats were matched in age (2·0-2·6 years), husbandry conditions and consumed ad libitum the same dry maintenance diet for at least 3 months prior to and 6 months following castration. All cats were castrated within 48 h of each other. All cats gained weight after castration. Mean BW was 4·67 (sd 0·70) kg at the start of the study and 5·93 (sd 1·38) kg at the end of the study, with individual weight gain ranging 3-53 % at 6 months post-neutering. The pre-conception BW of the queens of each cat was compared with the pre- and post-neutering BW of their offspring. The pre-conception BW of the queens was significantly correlated with the offspring's initial BW (ρ = 0·65, P = 0·01), final BW (ρ = 0·67, P = 0·01) and percentage BW change (ρ = 0·54, P = 0·04). A wide range of post-castration weight gains was observed among cats of similar backgrounds and housing conditions. Implementation of effective methods to control food consumption pre-conception and post-neutering may be a strategy for preventing obesity and obesity-related disorders in cats.

The initial cause of post-neutering weight gain in male cats is not entirely known. There is evidence that energy intake (EI) increases rapidly post-neutering, but it is not clear if neutering also decreases energy expenditure (EE) prior to weight gain. Thus, the purpose of this study was to determine if a decrease in EE contributes to the initial shift toward positive energy balance in neutered male cats. To determine the influence of neutering on EE independent of changes in EI and body weight (BW), male cats were fed at their pre-neutering maintenance EI and EE was measured at 4 days pre-neutering, 3-4 days post-neutering, and 9 days post- neutering. Ad libitum food access was then provided for 6 months. Body composition was measured and blood samples collected for serum chemistry at pre-neutering and 7 days, 13 days and 6 months post-neutering. Total energy expenditure (TEE) adjusted for lean body mass (LBM) did not change in cats from pre-neutering to 9 days post-neutering. However, TEE adjusted for BW and resting energy expenditure adjusted for either LBM or BW showed a small, but significant (P

AimsThe purpose of the study was to establish if enzyme activities from key metabolic pathways and levels of markers of oxidative damage to proteins and lipids differed between distinct liver mitochondrial sub-populations, and which specific sub-populations contributed to these differences.Main methodsMale C57BL/6J mice were fed non-purified diet for one month then separated into two groups, control and calorie-restricted (CR). The two groups were fed semi-purified diet (AIN93G), with the CR group receiving 40% less calories than controls. After two months, enzyme activities and markers of oxidative damage in mitochondria were determined.Key findingsIn all mitochondrial sub-populations, enzyme activities and markers of oxidative damage, from control and CR groups, showed a pattern of M1>M3>M10. Higher acyl-CoA dehydrogenase (β-oxidation) and β-hydroxybutyrate dehydrogenase (ketogenesis) activities and lower carbonyl and TBARS levels were observed in M1 and M3 fractions from CR mice. ETC enzyme activities did not show a consistent pattern. In the Krebs cycle, citrate synthase and aconitase activities decreased while succinate dehydrogenase and malate dehydrogenase activities increased in the M1 mitochondria from the CR versus control mice.SignificanceCR does not produce uniform changes in enzyme activities or markers of oxidative damage in mitochondrial sub-populations, with changes occurring primarily in the heavy mitochondrial populations. Centrifugation at 10,000 g to isolate mitochondria likely dilutes the mitochondrial populations which show the greatest response to CR. Use of lower centrifugal force (3000 g or lower) may be beneficial for some studies.

To investigate the role mitochondrial membrane lipids play in the actions of CR (calorie restriction), C57BL/6 mice were assigned to four groups (control and three 40% CR groups) and the CR groups were fed diets containing soya bean oil (also in the control diet), fish oil or lard. The fatty acid composition of the major mitochondrial phospholipid classes, proton leak and H(2)O(2) production were measured in liver mitochondria following 1 month of CR. The results indicate that mitochondrial phospholipid fatty acids reflect the PUFA (polyunsaturated fatty acid) profile of the dietary lipid sources. CR significantly decreased the capacity of ROS (reactive oxygen species) production by Complex III but did not markedly alter proton leak and ETC (electron transport chain) enzyme activities. Within the CR regimens, the CR-fish group had decreased ROS production by both Complexes I and III, and increased proton leak when compared with the other CR groups. The CR-lard group showed the lowest proton leak compared with the other CR groups. The ETC enzyme activity measurements in the CR regimens showed that Complex I activity was decreased in both the CR-fish and CR-lard groups. Moreover, the CR-fish group also had lower Complex II activity compared with the other CR groups. These results indicate that dietary lipid composition does influence liver mitochondrial phospholipid composition, ROS production, proton leak and ETC enzyme activities in CR animals.

To investigate the role mitochondrial membrane lipids play in the actions of CR (calorie restriction), C57BL/6 mice were assigned to four groups (control and three 40% CR groups) and the CR groups were fed diets containing soya bean oil (also in the control diet), fish oil or lard. The fatty acid composition of the major mitochondrial phospholipid classes, proton leak and H(2)O(2) production were measured in liver mitochondria following 1 month of CR. The results indicate that mitochondrial phospholipid fatty acids reflect the PUFA (polyunsaturated fatty acid) profile of the dietary lipid sources. CR significantly decreased the capacity of ROS (reactive oxygen species) production by Complex III but did not markedly alter proton leak and ETC (electron transport chain) enzyme activities. Within the CR regimens, the CR-fish group had decreased ROS production by both Complexes I and III, and increased proton leak when compared with the other CR groups. The CR-lard group showed the lowest proton leak compared with the other CR groups. The ETC enzyme activity measurements in the CR regimens showed that Complex I activity was decreased in both the CR-fish and CR-lard groups. Moreover, the CR-fish group also had lower Complex II activity compared with the other CR groups. These results indicate that dietary lipid composition does influence liver mitochondrial phospholipid composition, ROS production, proton leak and ETC enzyme activities in CR animals.

The Src homology 2 domain-containing protein-tyrosine phosphatase Src homology phosphatase 2 (Shp2) is a negative regulator of hepatic insulin action in mice fed regular chow. To investigate the role of hepatic Shp2 in lipid metabolism and energy balance, we determined the metabolic effects of its deletion in mice challenged with a high-fat diet (HFD). We analyzed body mass, lipid metabolism, insulin sensitivity, and glucose tolerance in liver-specific Shp2-deficient mice (referred to herein as LSHKO) and control mice fed HFD. Hepatic Shp2 protein expression is regulated by nutritional status, increasing in mice fed HFD and decreasing during fasting. LSHKO mice gained less weight and exhibited increased energy expenditure compared with control mice. In addition, hepatic Shp2 deficiency led to decreased liver steatosis, enhanced insulin-induced suppression of hepatic glucose production, and impeded the development of insulin resistance after high-fat feeding. At the molecular level, LSHKO exhibited decreased hepatic endoplasmic reticulum stress and inflammation compared with control mice. In addition, tyrosine and serine phosphorylation of total and mitochondrial signal transducer and activator of transcription 3 were enhanced in LSHKO compared with control mice. In line with this observation and the increased energy expenditure of LSHKO, oxygen consumption rate was higher in liver mitochondria of LSHKO compared with controls. Collectively, these studies identify hepatic Shp2 as a novel regulator of systemic energy balance under conditions of high-fat feeding.

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

The polyunsaturated nature of n-3 fatty acids makes them prone to oxidative damage. However, it is not clear if n-3 fatty acids are simply a passive site for oxidative attack or if they also modulate mitochondrial reactive oxygen species (ROS) production. The present study used fat-1 transgenic mice, that are capable of synthesizing n-3 fatty acids, to investigate the influence of increases in n-3 fatty acids and resultant decreases in the n-6:n-3 ratio on liver mitochondrial H(2)O(2) production and electron transport chain (ETC) activity. There was an increase in n-3 fatty acids and a decrease in the n-6:n-3 ratio in liver mitochondria from the fat-1 compared to control mice. This change was largely due to alterations in the fatty acid composition of phosphatidylcholine and phosphatidylethanolamine, with only a small percentage of fatty acids in cardiolipin being altered in the fat-1 animals. The lipid changes in the fat-1 mice were associated with a decrease (p

Cats require more dietary protein than noncarnivorous species. Earlier work showed that cats lack the ability to regulate hepatic urea cycle enzymes in response to dietary protein concentration. We thus hypothesized that cats are unable to fully adapt protein oxidation to protein intake, particularly at low-protein concentrations. We used indirect respiration calorimetry to assess cats' ability to adapt substrate oxidation to diets containing different concentrations of protein, including 1 below their protein requirement. Nine cats (5 males and 4 females; 2.7 [+ or -] 0.5 y; 4.49 [+ or -] 0.19 kg) consumed each of 4 semipurified diets containing 7.5% [low protein (L[P.sup.3])], 14.2% [adequate protein (AP)], 27.1% [moderate protein (MP)], and 49.6% [high protein (HP)] of metabolizable energy from protein in a modified crossover design, beginning with the MP diet and then consuming the remaining diets in random order. After adaptation to each diet, cats completed a 5-d nitrogen balance trial and at least 2 12-h indirect calorimetry measurements. There was a significant effect of diet on protein oxidation (P< 0.0001), which measured 10.4 [+ or -] 0.5, 14.1 [+ or -] 1.0, 25.0 [+ or -] 1.7, and 53.2 [+ or -] 1.7% of total energy expenditure for the LP, AP, M,P and HP diets, respectively. The ratio of protein oxidation:protein intake was higher with the LP diet (1.39 [+ or -] 0.07) than the other 3 diets (AP, 1.00 [+ or -] 0.07; MP, 0.93 + 0.06; HP, 1.07 [+ or -] 0.03; P < 0.0001), indicating a net loss of protein with the LP diet. Thus, cats are able to adapt protein oxidation to a wide range of dietary protein concentrations, provided their minimum protein requirement is met.

Dietary energy restriction (ER) is used to treat obesity in cats but it is often unsuccessful. The purpose of this study was to determine whether ER results in a sustained decrease in mass-adjusted energy expenditure (EE) that may oppose weight loss and promote weight regain. EE and body composition were measured in 10 adult neutered cats at 3 time points: baseline (obese cats), during weight loss (40% ER), and following weight regain. The cats started with a body weight (BW) of 6.1 [+ or -] 0.30 kg, body condition score (BCS) of 7.6 [+ or -] 0.14 (on a 9-point scale), and fat body mass (FM) of 38 [+ or -] 1.0% of BW. After weight loss, BWwas 5.0 [+ or -] 0.19 kg, BCS was 5.5 [+ or -] 0.07 kg, and FM was 31 [+ or -] 1.6% (P< 0.01). After weight regain, BWwas 6.2 [+ or -] 0.30 kg, BCS was 7.7 [+ or -] 0.16, and FM was 42 [+ or -] 1.8% (P< 0.01). Total EE decreased from 1258 [+ or -] 33.7 kJ/d to 1025 [+ or -] 39.6 kJ/d during weight loss (P < 0.001). After weight regain, EE was still lower than baseline (1103 [+ or -] 41.5 k J/d, P < 0.001). Energy intake (El) at baseline (1337 [+ or -] 50.6 k J/d) was higher than El after weight loss and regain (1217 [+ or -] 61.2 k J/d), resulting in no differences in energy balance (78 [+ or -] 30.4 and 104 [+ or -] 35.4 k J/d, respectively, P = 0.581). These results support the hypothesis that ER results in a mass-adjusted decrease in EE in cats that is maintained after weight regain.

Caloric restriction (CR) is known to retard the aging process, and a marker of aging is decreased energy expenditure (EE). To assess longitudinal effects of CR on EE in rhesus monkeys (Macaca mulatta), data from 41 males (M) and 26 females (F) subjected to 9 or 15 yr of CR were studied. EE and body composition of monkeys 11-28 yr of age were measured using indirect calorimetry and dual X-ray absorptiometry. Total EE (24-h EE) was divided into daytime (day EE), nighttime (night EE), and daytime minus nighttime (D--NEE). M calorie-restricted monkeys showed a lower 24-h EE (means [+ or -] SD = 568 [+ or -] 96 kcal/day, P < 0.0001) than controls (C; 630 [+ or -] 129 kcal/day). Calorie-restricted M had a lower night EE (difference = 36 kcal P < 0.0001) compared with C M, but after adjusting for FFM and FM, night EE was not different between calorie-restricted and C males (P = 0.72). The 24-b EE decreased with age (13 kcal decrease/yr, P < 0.0001), but there was no difference between CR and C. Adjusted for FFM and FM, D--N EE decreased with age (9 kcal/yr, P < 0.0001), with no interaction with age (P = 0.72). The F were compared with age-matched M selected from the male cohort. F had a lower 24-h EE (496 [+ or -] 84 kcal/day) than M (636 [+ or -] 139 kcal/day) (P < 0.0001). Adjusting for FFM and FM, night EE was lower in F compared with M (difference = 18 kcal, P = 0.077). Night EE did not differ between calorie-restricted and C younger monkeys after adjusting for FFM and FM. In conclusion, CR did not alter the age-related decrease in EE with CR. metabolic rate; dietary restriction; indirect calorimetry