Isolation of Progenitors that Exhibit Myogenic/Osteogenic Bipotency In Vitro by Fluorescence-Activated Cell Sorting from Human Fetal Muscle

Fluorescence-activated cell sorting (FACS) strategies to purify distinct cell types from the pool of fetal human myofiber-associated (hMFA) cells were developed. We demonstrate that cells expressing the satellite cell marker PAX7 are highly enriched within the subset of CD45−CD11b−GlyA−CD31−CD34−CD56intITGA7hi hMFA cells. These CD45−CD11b−GlyA−CD31−CD34−CD56intITGA7hi cells lack adipogenic capacity but exhibit robust, bipotent myogenic and osteogenic activity in vitro and engraft myofibers when transplanted into mouse muscle. In contrast, CD45−CD11b−GlyA−CD31−CD34+ fetal hMFA cells represent stromal constituents of muscle that do not express PAX7, lack myogenic function, and exhibit adipogenic and osteogenic capacity in vitro. Adult muscle likewise contains PAX7+ CD45−CD11b−GlyA−CD31−CD34−CD56intITGA7hi hMFA cells with in vitro myogenic and osteogenic activity, although these cells are present at lower frequency in comparison to their fetal counterparts. The ability to directly isolate functionally distinct progenitor cells from human muscle will enable novel insights into muscle lineage specification and homeostasis.Stem Cell and Regenerative Biolog

The cold-induced lipokine 12,13-diHOME promotes fatty acid transport into brown adipose tissue

Brown adipose tissue (BAT) and beige adipose tissue combust fuels for heat production in adult humans, and so constitute an appealing target for the treatment of metabolic disorders such as obesity, diabetes and hyperlipidemia1,2. Cold exposure can enhance energy expenditure by activating BAT, and it has been shown to improve nutrient metabolism3–5. These therapies, however, are time consuming and uncomfortable, demonstrating the need for pharmacological interventions. Recently, lipids have been identified that are released from tissues and act locally or systemically to promote insulin sensitivity and glucose tolerance; as a class, these lipids are referred to as ‘lipokines’6–8. Because BAT is a specialized metabolic tissue that takes up and burns lipids and is linked to systemic metabolic homeostasis, we hypothesized that there might be thermogenic lipokines that activate BAT in response to cold. Here we show that the lipid 12,13-dihydroxy-9Z-octadecenoic acid (12,13-diHOME) is a stimulator of BAT activity, and that its levels are negatively correlated with body-mass index and insulin sensitivity. Using a global lipidomic analysis, we found that 12,13-diHOME was increased in the circulation of humans and mice exposed to cold. Furthermore, we found that the enzymes that produce 12,13-diHOME were uniquely induced in BAT by cold stimulation. The injection of 12,13-diHOME acutely activated BAT fuel uptake and enhanced cold tolerance, which resulted in decreased levels of serum triglycerides. Mechanistically, 12,13-diHOME increased fatty acid (FA) uptake into brown adipocytes by promoting the translocation of the FA transporters FATP1 and CD36 to the cell membrane. These data suggest that 12,13-diHOME, or a functional analog, could be developed as a treatment for metabolic disorders

Metallothionein (MT) -I and MT-II Expression Are Induced and Cause Zinc Sequestration in the Liver after Brain Injury

Experiments with transgenic over-expressing, and null mutant mice have determined that metallothionein-I and -II (MT-I/II) are protective after brain injury. MT-I/II is primarily a zinc-binding protein and it is not known how it provides neuroprotection to the injured brain or where MT-I/II acts to have its effects. MT-I/II is often expressed in the liver under stressful conditions but to date, measurement of MT-I/II expression after brain injury has focused primarily on the injured brain itself. In the present study we measured MT-I/II expression in the liver of mice after cryolesion brain injury by quantitative reverse-transcriptase PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) with the UC1MT antibody. Displacement curves constructed using MT-I/II knockout (MT-I/II−/−) mouse tissues were used to validate the ELISA. Hepatic MT-I and MT-II mRNA levels were significantly increased within 24 hours of brain injury but hepatic MT-I/II protein levels were not significantly increased until 3 days post injury (DPI) and were maximal at the end of the experimental period, 7 DPI. Hepatic zinc content was measured by atomic absorption spectroscopy and was found to decrease at 1 and 3 DPI but returned to normal by 7DPI. Zinc in the livers of MT-I/II−/− mice did not show a return to normal at 7 DPI which suggests that after brain injury, MT-I/II is responsible for sequestering elevated levels of zinc to the liver. Conclusion: MT-I/II is up-regulated in the liver after brain injury and modulates the amount of zinc that is sequestered to the liver

Telomerase Reverse Transcriptase Expression Marks a Population of Rare Adipose Tissue Stem Cells.

In adult tissues such as adipose tissue, post-mitotic cells like adipocytes can be replaced by differentiation of a population of tissue-resident stem cells. Expression of mouse telomerase reverse transcriptase (mTert) is a hallmark of stem cell populations, and previous efforts to identify tissue-resident adult stem cells by measuring mTert expression have increased our understanding of stem cell biology significantly. Here, we used a doxycycline-inducible mouse model to perform longitudinal, live-animal lineage-tracing of mTert-expressing cells for more than 1 year. We identified a rare

Silk Hydrogel-Mediated Delivery of Bone Morphogenetic Protein 7 Directly to Subcutaneous White Adipose Tissue Increases Browning and Energy Expenditure

Increasing the mass and/or activity of brown adipose tissue (BAT) is one promising avenue for treating obesity and related metabolic conditions, given that BAT has a high potential for energy expenditure and is capable of improving glucose and lipid homeostasis. BAT occurs either in discrete classical depots, or interspersed in white adipose tissue (WAT), termed inducible/recruitable BAT, or \u27beige/brite\u27 adipocytes. We and others have demonstrated that bone morphogenetic protein 7 (BMP7) induces brown adipogenesis in committed and uncommitted progenitor cells, resulting in increased energy expenditure and reduced weight gain in mice. BMP7 is therefore a reliable growth factor to induce browning of WAT. In this study, we sought to deliver BMP7 specifically to subcutaneous (sc)WAT in order to induce tissue-resident progenitor cells to differentiate into energy-expending recruitable brown adipocytes, without off-target effects like bone formation, which can occur when BMPs are in the presence of bone progenitor cells (outside of WAT). BMP7 delivery directly to WAT may also promote tissue innervation, or directly activate mitochondrial activity in brown adipocytes, as we have demonstrated previously. We utilized silk protein in the form of an injectable hydrogel carrying BMP7. Silk scaffolds are useful for delivery of substances due to favorable material properties, including controlled release of therapeutic proteins in an active form, biocompatibility with minimal immunogenic response, and prior FDA approval for some medical materials. For this study, the silk was engineered to meet desirable release kinetics for BMP7 in order to mimic our prior brown adipocyte differentiation studies. Fluorescently-labeled silk hydrogel loaded with BMP7 was directly injected into WAT through the skin and monitored by non-invasive whole body imaging, including in UCP1-luciferase reporter mice, thereby enabling an approach that is translatable to humans. Injection of the BMP7-loaded silk hydrogels into the subcutaneous WAT of mice resulted in browning , including the development of multilocular, uncoupling protein 1 (UCP1)-positive brown adipocytes, and an increase in whole-body energy expenditure and skin temperature. In diet-induced obese mice, BMP7-loaded silk delivery to subcutaneous WAT resulted in less weight gain, reduced circulating glucose and lower respiratory exchange ratio (RER). In summary, BMP7 delivery via silk scaffolds directly into scWAT is a novel translational approach to increase browning and energy expenditure, and represents a potential therapeutic avenue for delivering substances directly to adipose depots in pursuit of metabolic treatments

Brown adipose tissue thermogenic adaptation requires Nrf1-mediated proteasomal activity

Objective Brown adipose tissue (BAT) generates heat in response to cold, and low BAT activity has been linked to obesity. However, recent studies were inconclusive as to whether BAT is involved in diet‐induced thermogenesis and mitigates weight gain from prolonged overeating. Therefore, this study investigated whether BAT activity is related to metabolic adaptation arising from 8 weeks of overfeeding in humans. Methods Fourteen men (aged 24 ± 3 years, BMI 24.5 ± 1.6 kg/m2) were overfed by 40% for 8 weeks. Before and after, energy expenditure and metabolic adaptation were measured by whole‐room respiratory calorimetry. A marker of BAT activity was measured using infrared imaging of the supraclavicular BAT depot. Results At the end of 8 weeks of overfeeding, metabolic adaptation—defined as the percent increase in sleeping energy expenditure beyond that expected from weight gain—rose from −0.9 ± 3.9% to 4.7 ± 5.6% (P = 0.001). However, BAT thermal activity was unchanged (P = 0.81). Moreover, BAT thermal activity did not correlate with the degree of metabolic adaptation (P = 0.32) or with the change in body weight (P = 0.51). Conclusions BAT thermal activity does not change in response to overfeeding, nor does it correlate with adaptive thermogenesis. Our data suggest that BAT does not mediate metabolic adaptation to overeating in humans

Unraveling the functional dark matter through global metagenomics

Metagenomes encode an enormous diversity of proteins, reflecting a multiplicity of functions and activities1,2. Exploration of this vast sequence space has been limited to a comparative analysis against reference microbial genomes and protein families derived from those genomes. Here, to examine the scale of yet untapped functional diversity beyond what is currently possible through the lens of reference genomes, we develop a computational approach to generate reference-free protein families from the sequence space in metagenomes. We analyse 26,931 metagenomes and identify 1.17 billion protein sequences longer than 35 amino acids with no similarity to any sequences from 102,491 reference genomes or the Pfam database3. Using massively parallel graph-based clustering, we group these proteins into 106,198 novel sequence clusters with more than 100 members, doubling the number of protein families obtained from the reference genomes clustered using the same approach. We annotate these families on the basis of their taxonomic, habitat, geographical and gene neighbourhood distributions and, where sufficient sequence diversity is available, predict protein three-dimensional models, revealing novel structures. Overall, our results uncover an enormously diverse functional space, highlighting the importance of further exploring the microbial functional dark matter