Metabolic plasticity and obesity-associated changes in diurnal postexercise metabolism in mice.

BACKGROUND: Circadian disruption is widespread and increases the risk of obesity. Timing of therapeutic interventions may promote coherent and efficient gating of metabolic processes and restore energy homeostasis. AIM: To characterize the diurnal postexercise metabolic state in mice and to identify the influence of diet-induced obesity on identified outcomes. METHODS: C57BL6/NTac male mice (6 wks of age) were fed a standard chow or high-fat diet for 5 weeks. At week 5, mice were subjected to a 60-min (16 m/min, 5 % incline) running bout (or sham) during the early rest (day) or early active (night) phase. Tissue and serum samples were collected immediately post-exercise (n = 6/group). In vivo glucose oxidation was measured after oral administration of 13C-glucose via 13CO2 exhalation analysis in metabolic cages. Basal and isoproterenol-stimulated adipose tissue lipolysis was assessed ex vivo for 1 h following exercise. RESULTS: Lean mice displayed exercise-timing-specific plasticity in metabolic outcomes, including phase-specificity in systemic glucose metabolism and adipose-tissue-autonomous lipolytic activity depending on time of day. Conversely, obesity impaired temporal postexercise differences in whole-body glucose oxidation, as well as the phase- and exercise-mediated induction of lipolysis in isolated adipose tissue. This obesity-induced alteration in diurnal metabolism, as well as the indistinct response to exercise, was observed concomitant with disruption of core clock gene expression in peripheral tissues. CONCLUSIONS: Overall, high-fat fed obese mice exhibit metabolic inflexibility, which is also evident in the diurnal exercise response. Our study provides physiological insight into exercise timing-dependent aspects in the dynamic regulation of metabolism and the influence of obesity on this biology.

Trigonelline is an NAD+ precursor that improves muscle function during ageing and is reduced in human sarcopenia.

Mitochondrial dysfunction and low nicotinamide adenine dinucleotide (NAD+) levels are hallmarks of skeletal muscle ageing and sarcopenia1-3, but it is unclear whether these defects result from local changes or can be mediated by systemic or dietary cues. Here we report a functional link between circulating levels of the natural alkaloid trigonelline, which is structurally related to nicotinic acid4, NAD+ levels and muscle health in multiple species. In humans, serum trigonelline levels are reduced with sarcopenia and correlate positively with muscle strength and mitochondrial oxidative phosphorylation in skeletal muscle. Using naturally occurring and isotopically labelled trigonelline, we demonstrate that trigonelline incorporates into the NAD+ pool and increases NAD+ levels in Caenorhabditis elegans, mice and primary myotubes from healthy individuals and individuals with sarcopenia. Mechanistically, trigonelline does not activate GPR109A but is metabolized via the nicotinate phosphoribosyltransferase/Preiss-Handler pathway5,6 across models. In C. elegans, trigonelline improves mitochondrial respiration and biogenesis, reduces age-related muscle wasting and increases lifespan and mobility through an NAD+-dependent mechanism requiring sirtuin. Dietary trigonelline supplementation in male mice enhances muscle strength and prevents fatigue during ageing. Collectively, we identify nutritional supplementation of trigonelline as an NAD+-boosting strategy with therapeutic potential for age-associated muscle decline.

Seasonal light hours modulate peripheral clocks and energy metabolism in mice.

Except for latitudes close to the equator, seasonal variation in light hours can change dramatically between summer and winter. Yet investigations into the interplay between energy metabolism and circadian rhythms typically use a 12 h light:12 h dark photoperiod corresponding to the light duration at the equator. We hypothesized that altering the seasonal photoperiod affects both the rhythmicity of peripheral tissue clocks and energy homeostasis. Mice were housed at photoperiods representing either light hours in summer, winter, or the equinox. Mice housed at a winter photoperiod exhibited an increase in the amplitude of rhythmic lipid metabolism and a modest reduction in fat mass and liver triglyceride content. Comparing melatonin-proficient and -deficient mice, the effect of seasonal light on energy metabolism was largely driven by differences in the rhythmicity of food intake and not melatonin. Together, these data indicate that seasonal light impacts energy metabolism by modulating the timing of eating.

Milk oligosaccharide-driven persistence of Bifidobacterium pseudocatenulatum modulates local and systemic microbial metabolites upon synbiotic treatment in conventionally colonized mice.

BACKGROUND: Bifidobacteria represent an important gut commensal in humans, particularly during initial microbiome assembly in the first year of life. Enrichment of Bifidobacterium is mediated though the utilization of human milk oligosaccharides (HMOs), as several human-adapted species have dedicated genomic loci for transport and metabolism of these glycans. This results in the release of fermentation products into the gut lumen which may offer physiological benefits to the host. Synbiotic pairing of probiotic species with a cognate prebiotic delivers a competitive advantage, as the prebiotic provides a nutrient niche. METHODS: To determine the fitness advantage and metabolic characteristics of an HMO-catabolizing Bifidobacterium strain in the presence or absence of 2'-fucosyllactose (2'-FL), conventionally colonized mice were gavaged with either Bifidobacterium pseudocatenulatum MP80 (B.p. MP80) (as the probiotic) or saline during the first 3 days of the experiment and received water or water containing 2'-FL (as the prebiotic) throughout the study. RESULTS: 16S rRNA gene sequencing revealed that mice provided only B.p. MP80 were observed to have a similar microbiota composition as control mice throughout the experiment with a consistently low proportion of Bifidobacteriaceae present. Using 1H NMR spectroscopy, similar metabolic profiles of gut luminal contents and serum were observed between the control and B.p. MP80 group. Conversely, synbiotic supplemented mice exhibited dramatic shifts in their community structure across time with an overall increased, yet variable, proportion of Bifidobacteriaceae following oral inoculation. Parsing the synbiotic group into high and moderate bifidobacterial persistence based on the median proportion of Bifidobacteriaceae, significant differences in gut microbial diversity and metabolite profiles were observed. Notably, metabolites associated with the fermentation of 2'-FL by bifidobacteria were significantly greater in mice with a high proportion of Bifidobacteriaceae in the gut suggesting metabolite production scales with population density. Moreover, 1,2-propanediol, a fucose fermentation product, was only observed in the liver and brain of mice harboring high proportions of Bifidobacteriaceae. CONCLUSIONS: This study reinforces that the colonization of the gut with a commensal microorganism does not guarantee a specific functional output. Video Abstract.

Time of day determines postexercise metabolism in mouse adipose tissue.

The circadian clock is a cell-autonomous transcription-translation feedback mechanism that anticipates and adapts physiology and behavior to different phases of the day. A variety of factors including hormones, temperature, food-intake, and exercise can act on tissue-specific peripheral clocks to alter the expression of genes that influence metabolism, all in a time-of-day dependent manner. The aim of this study was to elucidate the effects of exercise timing on adipose tissue metabolism. We performed RNA sequencing on inguinal adipose tissue of mice immediately following maximal exercise or sham treatment at the early rest or early active phase. Only during the early active phase did exercise elicit an immediate increase in serum nonesterified fatty acids. Furthermore, early active phase exercise increased expression of markers of thermogenesis and mitochondrial proliferation in inguinal adipose tissue. In vitro, synchronized 3T3-L1 adipocytes showed a timing-dependent difference in Adrb2 expression, as well as a greater lipolytic activity. Thus, the response of adipose tissue to exercise is time-of-day sensitive and may be partly driven by the circadian clock. To determine the influence of feeding state on the time-of-day response to exercise, we replicated the experiment in 10-h-fasted early rest phase mice to mimic the early active phase metabolic status. A 10-h fast led to a similar lipolytic response as observed after active phase exercise but did not replicate the transcriptomic response, suggesting that the observed changes in gene expression are not driven by feeding status. In conclusion, acute exercise elicits timing-specific effects on adipose tissue to maintain metabolic homeostasis.

Protocol to assess arteriovenous differences across the liver and hindlimb muscles in mice following treadmill exercise.

The tissue-specific release and uptake of metabolites in response to exercise is incompletely understood. Here, we detail a protocol to assess arteriovenous differences across the liver and hindlimb muscles in response to treadmill exercise in mice. We describe steps for the treadmill running of mice and the region-specific sampling of blood from the liver and hindlimb. This procedure is particularly relevant for the study of tissue-specific metabolism in response to exercise. For complete details on the use and execution of this protocol, please refer to Sato et al. (2022).1.

Atlas of exercise metabolism reveals time-dependent signatures of metabolic homeostasis.

Tissue sensitivity and response to exercise vary according to the time of day and alignment of circadian clocks, but the optimal exercise time to elicit a desired metabolic outcome is not fully defined. To understand how tissues independently and collectively respond to timed exercise, we applied a systems biology approach. We mapped and compared global metabolite responses of seven different mouse tissues and serum after an acute exercise bout performed at different times of the day. Comparative analyses of intra- and inter-tissue metabolite dynamics, including temporal profiling and blood sampling across liver and hindlimb muscles, uncovered an unbiased view of local and systemic metabolic responses to exercise unique to time of day. This comprehensive atlas of exercise metabolism provides clarity and physiological context regarding the production and distribution of canonical and novel time-dependent exerkine metabolites, such as 2-hydroxybutyrate (2-HB), and reveals insight into the health-promoting benefits of exercise on metabolism.

Bifidobacterium catabolism of human milk oligosaccharides overrides endogenous competitive exclusion driving colonization and protection.

Understanding how exogenous microbes stably colonize the animal gut is essential to reveal mechanisms of action and tailor effective probiotic treatments. Bifidobacterium species are naturally enriched in the gastrointestinal tract of breast-fed infants. Human milk oligosaccharides (HMOs) are associated with this enrichment. However, direct mechanistic proof of the importance of HMOs in this colonization is lacking given milk contains additional factors that impact the gut microbiota. This study examined mice supplemented with the HMO 2'fucosyllactose (2'FL) together with a 2'FL-consuming strain, Bifidobacterium pseudocatenulatum MP80. 2'FL supplementation creates a niche for high levels of B.p. MP80 persistence, similar to Bifidobacterium levels seen in breast-fed infants. This synergism impacted gut microbiota composition, activated anti-inflammatory pathways and protected against chemically-induced colitis. These results demonstrate that bacterial-milk glycan interactions alone drive enrichment of beneficial Bifidobacterium and provide a model for tunable colonization thus facilitating insight into mechanisms of health promotion by bifidobacteriain neonates.

Bifidobacteria-mediated immune system imprinting early in life.

Immune-microbe interactions early in life influence the risk of allergies, asthma, and other inflammatory diseases. Breastfeeding guides healthier immune-microbe relationships by providing nutrients to specialized microbes that in turn benefit the host's immune system. Such bacteria have co-evolved with humans but are now increasingly rare in modern societies. Here we show that a lack of bifidobacteria, and in particular depletion of genes required for human milk oligosaccharide (HMO) utilization from the metagenome, is associated with systemic inflammation and immune dysregulation early in life. In breastfed infants given Bifidobacterium infantis EVC001, which expresses all HMO-utilization genes, intestinal T helper 2 (Th2) and Th17 cytokines were silenced and interferon ß (IFNß) was induced. Fecal water from EVC001-supplemented infants contains abundant indolelactate and B. infantis-derived indole-3-lactic acid (ILA) upregulated immunoregulatory galectin-1 in Th2 and Th17 cells during polarization, providing a functional link between beneficial microbes and immunoregulation during the first months of life.

Nampt controls skeletal muscle development by maintaining Ca2+ homeostasis and mitochondrial integrity.

OBJECTIVE: NAD+ is a co-factor and substrate for enzymes maintaining energy homeostasis. Nicotinamide phosphoribosyltransferase (NAMPT) controls NAD+ synthesis, and in skeletal muscle, NAD+ is essential for muscle integrity. However, the underlying molecular mechanisms by which NAD+ synthesis affects muscle health remain poorly understood. Thus, the objective of the current study was to delineate the role of NAMPT-mediated NAD+ biosynthesis in skeletal muscle development and function. METHODS: To determine the role of Nampt in muscle development and function, we generated skeletal muscle-specific Nampt KO (SMNKO) mice. We performed a comprehensive phenotypic characterization of the SMNKO mice, including metabolic measurements, histological examinations, and RNA sequencing analyses of skeletal muscle from SMNKO mice and WT littermates. RESULTS: SMNKO mice were smaller, with phenotypic changes in skeletal muscle, including reduced fiber area and increased number of centralized nuclei. The majority of SMNKO mice died prematurely. Transcriptomic analysis identified that the gene encoding the mitochondrial permeability transition pore (mPTP) regulator Cyclophilin D (Ppif) was upregulated in skeletal muscle of SMNKO mice from 2 weeks of age, with associated increased sensitivity of mitochondria to the Ca2+-stimulated mPTP opening. Treatment of SMNKO mice with the Cyclophilin D inhibitor, Cyclosporine A, increased membrane integrity, decreased the number of centralized nuclei, and increased survival. CONCLUSIONS: Our study demonstrates that NAMPT is crucial for maintaining cellular Ca2+ homeostasis and skeletal muscle development, which is vital for juvenile survival.

Compound- and fiber type-selective requirement of AMPKγ3 for insulin-independent glucose uptake in skeletal muscle.

OBJECTIVE: The metabolic master-switch AMP-activated protein kinase (AMPK) mediates insulin-independent glucose uptake in muscle and regulates the metabolic activity of brown and beige adipose tissue (BAT). The regulatory AMPKγ3 isoform is uniquely expressed in skeletal muscle and potentially in BAT. Herein, we investigated the role that AMPKγ3 plays in mediating skeletal muscle glucose uptake and whole-body glucose clearance in response to small-molecule activators that act on AMPK via distinct mechanisms. We also assessed whether γ3 plays a role in adipose thermogenesis and browning. METHODS: Global AMPKγ3 knockout (KO) mice were generated. A systematic whole-body, tissue, and molecular phenotyping linked to glucose homeostasis was performed in γ3 KO and wild-type (WT) mice. Glucose uptake in glycolytic and oxidative skeletal muscle ex vivo as well as blood glucose clearance in response to small molecule AMPK activators that target the nucleotide-binding domain of the γ subunit (AICAR) and allosteric drug and metabolite (ADaM) site located at the interface of the α and ß subunit (991, MK-8722) were assessed. Oxygen consumption, thermography, and molecular phenotyping with a ß3-adrenergic receptor agonist (CL-316,243) treatment were performed to assess BAT thermogenesis, characteristics, and function. RESULTS: Genetic ablation of γ3 did not affect body weight, body composition, physical activity, and parameters associated with glucose homeostasis under chow or high-fat diet. γ3 deficiency had no effect on fiber-type composition, mitochondrial content and components, or insulin-stimulated glucose uptake in skeletal muscle. Glycolytic muscles in γ3 KO mice showed a partial loss of AMPKα2 activity, which was associated with reduced levels of AMPKα2 and ß2 subunit isoforms. Notably, γ3 deficiency resulted in a selective loss of AICAR-, but not MK-8722-induced blood glucose-lowering in vivo and glucose uptake specifically in glycolytic muscle ex vivo. We detected γ3 in BAT and found that it preferentially interacts with α2 and ß2. We observed no differences in oxygen consumption, thermogenesis, morphology of BAT and inguinal white adipose tissue (iWAT), or markers of BAT activity between WT and γ3 KO mice. CONCLUSIONS: These results demonstrate that γ3 plays a key role in mediating AICAR- but not ADaM site binding drug-stimulated blood glucose clearance and glucose uptake specifically in glycolytic skeletal muscle. We also showed that γ3 is dispensable for ß3-adrenergic receptor agonist-induced thermogenesis and browning of iWAT.

Indole-3-lactic acid associated with Bifidobacterium-dominated microbiota significantly decreases inflammation in intestinal epithelial cells.

BACKGROUND: Bifidobacterium longum subsp. infantis (B. infantis) is a commensal bacterium that colonizes the gastrointestinal tract of breast-fed infants. B. infantis can efficiently utilize the abundant supply of oligosaccharides found in human milk (HMO) to help establish residence. We hypothesized that metabolites from B. infantis grown on HMO produce a beneficial effect on the host. RESULTS: In a previous study, we demonstrated that B. infantis routinely dominated the fecal microbiota of a breast fed Bangladeshi infant cohort (1). Characterization of the fecal metabolome of binned samples representing high and low B. infantis populations from this cohort revealed higher amounts of the tryptophan metabolite indole-3-lactic acid (ILA) in feces with high levels of B. infantis. Further in vitro analysis confirmed that B. infantis produced significantly greater quantities of the ILA when grown on HMO versus lactose, suggesting a growth substrate relationship to ILA production. The direct effects of ILA were assessed in a macrophage cell line and intestinal epithelial cell lines. ILA (1-10 mM) significantly attenuated lipopolysaccharide (LPS)-induced activation of NF-kB in macrophages. ILA significantly attenuated TNF-α- and LPS-induced increase in the pro-inflammatory cytokine IL-8 in intestinal epithelial cells. ILA increased mRNA expression of the aryl hydrogen receptor (AhR)-target gene CYP1A1 and nuclear factor erythroid 2-related factor 2 (Nrf2)-targeted genes glutathione reductase 2 (GPX2), superoxide dismutase 2 (SOD2), and NAD(P) H dehydrogenase (NQO1). Pretreatment with either the AhR antagonist or Nrf-2 antagonist inhibited the response of ILA on downstream effectors. CONCLUSIONS: These findings suggest that ILA, a predominant metabolite from B. infantis grown on HMO and elevated in infant stool high in B. infantis, and protects gut epithelial cells in culture via activation of the AhR and Nrf2 pathway.

VDAC2 is required for truncated BID-induced mitochondrial apoptosis by recruiting BAK to the mitochondria.

Truncated BID (tBID), a proapoptotic BCL2 family protein, induces BAK/BAX-dependent release of cytochrome c and other mitochondrial intermembrane proteins to the cytosol to induce apoptosis. The voltage-dependent anion channels (VDACs) are the primary gates for solutes across the outer mitochondrial membrane (OMM); however, their role in apoptotic OMM permeabilization remains controversial. Here, we report that VDAC2(-/-) (V2(-/-)) mouse embryonic fibroblasts (MEFs) are virtually insensitive to tBID-induced OMM permeabilization and apoptosis, whereas VDAC1(-/-), VDAC3(-/-) and VDAC1(-/-)/VDAC3(-/-) MEFs respond normally to tBID. V2(-/-) MEFs regain tBID sensitivity after VDAC2 expression. Furthermore, V2(-/-) MEFs are deficient in mitochondrial BAK despite normal tBID-mitochondrial binding and BAX/BAK expression. tBID sensitivity of BAK(-/-) MEFs is also reduced, although not to the same extent as V2(-/-) MEFs, which might result from their strong overexpression of BAX. Indeed, addition of recombinant BAX also sensitized V2(-/-) MEFs to tBID. Thus, VDAC2 acts as a crucial component in mitochondrial apoptosis by allowing the mitochondrial recruitment of BAK, thereby controlling tBID-induced OMM permeabilization and cell death.