IntroductionMesenchymal stem cells (MSCs) comprise an adult population that resides in many organs and exhibits multiple functions and phenotypes upon in vitro culture; MSCs can be induced to differentiate into mesodermal cell lineages, 1,2 support and regulate hematopoiesis, [3][4][5][6][7] regulate the stem-cell niche, [8][9][10][11][12] and may participate in the repair of tissue damage inflicted by normal wear and tear, injury, or disease. [13][14][15][16] MSCs comprise 0.01% to 0.001% of the bone marrow (BM)-nucleated cells and are obtained by expansion of the BM, plastic-adherent cell fraction. 1,[17][18][19][20][21] Under certain physiologic or experimental conditions, MSCs can be induced to differentiate in vitro into cells of the mesodermal lineage, specifically to osteocytes, adipocytes, chondrocytes, myocytes, tenocytes, myocardiocytes, and hematopoietic supportive stroma. 1,17,19,22 MSCs are an attractive cell-based therapy tool for developmental defects; degenerating diseases; and bone, cartilage, muscle, and other mesodermal tissue injuries. [23][24][25][26][27][28][29][30] Toll-like receptors (TLRs) are a class of molecules first discovered to play a role in body development 31 and later found to play a role in body maintenance. [32][33][34][35][36] The TLR family has been shown to be of importance in the innate immune system for the recognition of pathogen-associated molecular patterns (PAMPs) by immune cells, initiating a primary response toward invading pathogens and recruitment of the adaptive immune response. 32,[37][38][39][40][41][42][43][44][45][46][47][48][49] TLRs can be activated not only by pathogen components, but also by mammalian endogenous molecules such as heat-shock proteins and extracellular matrix breakdown products. [50][51][52] In the steady state, during the generation of immune cells, as well as under pathologic conditions, there are intimate interactions between lymphocyte populations and the organ stroma mesenchyme. These interactions regulate cell growth and differentiation and control cell functions. It is possible therefore that lymphocytes and the stromal mesenchyme share regulatory mechanisms. To test this possibility we aimed, in the present study, to examine the expression and possible regulatory functions of TLRs in mesenchymal cells.We explored the expression of TLRs by MSCs, the response of MSCs to known TLR activators, and the ability of a TLR-2 ligand to regulate MSC proliferation and differentiation. We show here that cultured MSCs express TLR molecules 1 to 8, but not TLR-9. Activation of MSCs by TLR ligands induced interleukin-6 (IL-6) secretion and nuclear factor B (NF-B) nuclear translocation. Pam3Cys, a prototypic ligand for TLR-2, induced proliferation of MSCs and regulated their differentiation. Relatively little is known about the signals that regulate MSC proliferation, differentiation, and development. 53,54 Our findings suggest that TLR signaling may play a role in restraining MSC differentiation and thus promote MSC renewal. Materials and methods ...
Summary Circadian clocks play a major role in orchestrating daily physiology, and their disruption can evoke metabolic diseases such as fatty liver and obesity. To study the role of circadian clocks in lipid homeostasis, we performed an extensive lipidomic analysis of liver tissues from wild type and clock-disrupted mice, fed either ad libitum or night fed. To our surprise, a similar fraction of lipids (~17%) oscillated in both mouse strains, most notably triglycerides, but with completely different phases. Moreover, several master lipid regulators (e.g. PPARα) and enzymes involved in triglyceride metabolism retained their circadian expression in clock-disrupted mice. Nighttime restricted feeding shifted the phase of triglyceride accumulation and resulted in ~50% decrease in hepatic triglyceride levels in wild type mice. Our findings suggest that circadian clocks and feeding time dictate the phase and levels of hepatic triglyceride accumulation, however oscillations in triglycerides can persist in the absence of a functional clock.
Mitochondria are major suppliers of cellular energy through nutrients oxidation. Little is known about the mechanisms that enable mitochondria to cope with changes in nutrient supply and energy demand that naturally occur throughout the day. To address this question, we applied MS-based quantitative proteomics on isolated mitochondria from mice killed throughout the day and identified extensive oscillations in the mitochondrial proteome. Remarkably, the majority of cycling mitochondrial proteins peaked during the early light phase. We found that rate-limiting mitochondrial enzymes that process lipids and carbohydrates accumulate in a diurnal manner and are dependent on the clock proteins PER1/2. In this conjuncture, we uncovered daily oscillations in mitochondrial respiration that peak during different times of the day in response to different nutrients. Notably, the diurnal regulation of mitochondrial respiration was blunted in mice lacking PER1/2 or on a high-fat diet. We propose that PERIOD proteins optimize mitochondrial metabolism to daily changes in energy supply/demand and thereby, serve as a rheostat for mitochondrial nutrient utilization.M itochondria serve as major suppliers of cellular energy through nutrient oxidation. One of the major challenges that mitochondria face is the adaptation to changes in nutrient supply and energy demand. An inability of mitochondria to deal with altered nutrient environment is associated with metabolic diseases, such as diabetes and obesity (1, 2).Mitochondria oxidize carbohydrates and lipids to generate ATP by a process known as oxidative phosphorylation. Pyruvate and fatty acids are transported from the cytoplasm into the mitochondrial matrix, where they are catabolized into acetyl CoA. Pyruvate is converted to acetyl CoA through the action of the pyruvate dehydrogenase complex (PDC), whereas fatty acids are oxidized through a cycle of reactions that trim two carbons at a time, generating one molecule of acetyl CoA in each cycle [i.e., fatty acid oxidation (FAO)]. The acetyl groups are then fed into the Krebs cycle for additional degradation, and the process culminates with the transfer of acetyl-derived high-energy electrons along the respiratory chain.Mounting evidence suggests that circadian clocks orchestrate our daily physiology and metabolism (3-6). The mammalian circadian timing system consists of a central pacemaker in the brain that is entrained by daily light-dark cycles and synchronizes subsidiary oscillators in virtually all cells of the body, in part by driving rhythmic feeding behavior. The core clock molecular circuitry relies on interlocked transcription-translation feedback loops that generate daily oscillations of gene expression in cultured cells and living animals (7). Many transcriptomes (8-12) and more recently, several proteomics (13-15) and metabolomics studies (16-21) highlighted the pervasive circadian control of metabolism.Rest-activity and feeding-fasting cycles that naturally occur throughout the day impose pronounced changes in nutrient s...
Polyamines are essential polycations present in all living cells. Polyamine levels are maintained from the diet and de novo synthesis, and their decline with age is associated with various pathologies. Here we show that polyamine levels oscillate in a daily manner. Both clock- and feeding-dependent mechanisms regulate the daily accumulation of key enzymes in polyamine biosynthesis through rhythmic binding of BMAL1:CLOCK to conserved DNA elements. In turn, polyamines control the circadian period in cultured cells and animals by regulating the interaction between the core clock repressors PER2 and CRY1. Importantly, we found that the decline in polyamine levels with age in mice is associated with a longer circadian period that can be reversed upon polyamine supplementation in the diet. Our findings suggest a crosstalk between circadian clocks and polyamine biosynthesis and open new possibilities for nutritional interventions against the decay in clock's function with age.
Photoactivation of bioactive molecules allows manipulation of cellular processes with high spatiotemporal precision. The recent emergence of visible-light excitable photoprotecting groups has the potential to further expand the established utility of the photoactivation strategy in biological applications by offering higher tissue penetration, diminished phototoxicity, and compatibility with other light-dependent techniques. Nevertheless, a critical barrier to such applications remains the significant hydrophobicity of most visible-light excitable photocaging groups. Here, we find that applying the conventional 2,6-sulfonation to meso-methyl BODIPY photocages is incompatible with their photoreaction due to an increase in the excited state barrier for photorelease. We present a simple, remote sulfonation solution to BODIPY photocages that imparts water solubility and provides control over cellular permeability while retaining their favorable spectroscopic and photoreaction properties. Peripherally disulfonated BODIPY photocages are cell impermeable, making them useful for modulation of cell-surface receptors, while monosulfonated BODIPY retains the ability to cross the cellular membrane and can modulate intracellular targets. This new approach is generalizable for controlling BODIPY localization and was validated by sensitization of mammalian cells and neurons by visible-light photoactivation of signaling molecules.
Obesity and related morbidities pose a major health threat. Obesity is associated with increased blood concentrations of the anorexigenic hormone leptin; however, obese individuals are resistant to its anorexigenic effects. We examined the phenomenon of reduced leptin signaling in a high-fat diet-induced obesity model in mice. Obesity promoted matrix metalloproteinase-2 (Mmp-2) activation in the hypothalamus, which cleaved the leptin receptor's extracellular domain and impaired leptin-mediated signaling. Deletion of Mmp-2 restored leptin receptor expression and reduced circulating leptin concentrations in obese mice. Lentiviral delivery of short hairpin RNA to silence in the hypothalamus of wild-type mice prevented leptin receptor cleavage and reduced fat accumulation. In contrast, lentiviral delivery of in the hypothalamus of mice promoted leptin receptor cleavage and higher body weight. In a genetic mouse model of obesity, transduction of cleavage-resistant leptin receptor in the hypothalamus reduced the rate of weight gain compared to uninfected mice or mice infected with the wild-type receptor. Immunofluorescence analysis showed that astrocytes and agouti-related peptide neurons were responsible for Mmp-2 secretion in mice fed a high-fat diet. These results suggest a mechanism for leptin resistance through activation of Mmp-2 and subsequent cleavage of the extracellular domain of the leptin receptor.
Molecular-level understanding of body weight control is essential for combating obesity. We show that female mice lacking tyrosine phosphatase epsilon (RPTPe) are protected from weight gain induced by high-fat food, ovariectomy, or old age and exhibit increased whole-body energy expenditure and decreased adiposity. RPTPe-deficient mice, in particular males, exhibit improved glucose homeostasis. Female nonobese RPTPe-deficient mice are leptin hypersensitive and exhibit reduced circulating leptin concentrations, suggesting that RPTPe inhibits hypothalamic leptin signaling in vivo. Leptin hypersensitivity persists in aged, ovariectomized, and high-fat-fed RPTPe-deficient mice, indicating that RPTPe helps establish obesity-associated leptin resistance. RPTPe associates with and dephosphorylates JAK2, thereby downregulating leptin receptor signaling. Leptin stimulation induces phosphorylation of hypothalamic RPTPe at its C-terminal Y695, which drives RPTPe to downregulate JAK2. RPTPe is therefore an inhibitor of hypothalamic leptin signaling in vivo, and provides controlled negative-feedback regulation of this pathway following its activation.
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