Proteome and phosphoproteome analysis of brown adipocytes reveals that RICTOR loss dampens global insulin/AKT signaling

Stimulating brown adipose tissue (BAT) activity represents a promising therapy for overcoming metabolic diseases. mTORC2 is important for regulating BAT metabolism, but its downstream targets have not been fully characterized. In this study, we apply proteomics and phosphoproteomics to investigate the downstream effectors of mTORC2 in brown adipocytes. We compare wild-type controls to isogenic cells with an induced knockout of the mTORC2 subunit RICTOR (Rictor-iKO) by stimulating each with insulin for a 30-minute time course. In Rictor-iKO cells, we identify decreases to the abundance of glycolytic and de novo lipogenesis enzymes, and increases to mitochondrial proteins as well as a set of proteins known to increase upon interferon stimulation. We also observe significant differences to basal phosphorylation due to chronic RICTOR loss including decreased phosphorylation of the lipid droplet protein perilipin-1 in Rictor-iKO cells, suggesting that RICTOR could be involved with regulating basal lipolysis or droplet dynamics. Finally, we observe mild dampening of acute insulin signaling response in Rictor-iKO cells, and a subset of AKT substrates exhibiting statistically significant dependence on RICTOR.Fil: Entwisle, Samuel W.. University of Washington; Estados UnidosFil: Martinez Calejman, Camila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Valente, Anthony S.. University of Washington; Estados UnidosFil: Lawrence, Robert T.. University of Washington; Estados UnidosFil: Hung, Chien Min. University Of Massachussets. Medical School; Estados UnidosFil: Guertin, David A.. University Of Massachussets. Medical School; Estados UnidosFil: Villen, Judit. University of Washington; Estados Unido

mTORC2-AKT signaling to ATP-citrate lyase drives brown adipogenesis and de novo lipogenesis

mTORC2 phosphorylates AKT in a hydrophobic motif site that is a biomarker of insulin sensitivity. In brown adipocytes, mTORC2 regulates glucose and lipid metabolism, however the mechanism has been unclear because downstream AKT signaling appears unaffected by mTORC2 loss. Here, by applying immunoblotting, targeted phosphoproteomics and metabolite profiling, we identify ATP-citrate lyase (ACLY) as a distinctly mTORC2-sensitive AKT substrate in brown preadipocytes. mTORC2 appears dispensable for most other AKT actions examined, indicating a previously unappreciated selectivity in mTORC2-AKT signaling. Rescue experiments suggest brown preadipocytes require the mTORC2/AKT/ACLY pathway to induce PPAR-gamma and establish the epigenetic landscape during differentiation. Evidence in mature brown adipocytes also suggests mTORC2 acts through ACLY to increase carbohydrate response element binding protein (ChREBP) activity, histone acetylation, and gluco-lipogenic gene expression. Substrate utilization studies additionally implicate mTORC2 in promoting acetyl-CoA synthesis from acetate through acetyl-CoA synthetase 2 (ACSS2). These data suggest that a principal mTORC2 action is controlling nuclear-cytoplasmic acetyl-CoA synthesis

Emergence of good conduct, scaling and Zipf laws in human behavioral sequences in an online world

We study behavioral action sequences of players in a massive multiplayer online game. In their virtual life players use eight basic actions which allow them to interact with each other. These actions are communication, trade, establishing or breaking friendships and enmities, attack, and punishment. We measure the probabilities for these actions conditional on previous taken and received actions and find a dramatic increase of negative behavior immediately after receiving negative actions. Similarly, positive behavior is intensified by receiving positive actions. We observe a tendency towards anti-persistence in communication sequences. Classifying actions as positive (good) and negative (bad) allows us to define binary 'world lines' of lives of individuals. Positive and negative actions are persistent and occur in clusters, indicated by large scaling exponents alpha~0.87 of the mean square displacement of the world lines. For all eight action types we find strong signs for high levels of repetitiveness, especially for negative actions. We partition behavioral sequences into segments of length n (behavioral `words' and 'motifs') and study their statistical properties. We find two approximate power laws in the word ranking distribution, one with an exponent of kappa-1 for the ranks up to 100, and another with a lower exponent for higher ranks. The Shannon n-tuple redundancy yields large values and increases in terms of word length, further underscoring the non-trivial statistical properties of behavioral sequences. On the collective, societal level the timeseries of particular actions per day can be understood by a simple mean-reverting log-normal model.Comment: 6 pages, 5 figure

PKC downregulation upon rapamycin treatment attenuates mitochondrial disease

Published in final edited form as: Nat Metab. 2020 December ; 2(12): 1472–1481. doi:10.1038/s42255-020-00319-x.Leigh syndrome is a fatal neurometabolic disorder caused by defects in mitochondrial function. Mechanistic target of rapamycin (mTOR) inhibition with rapamycin attenuates disease progression in a mouse model of Leigh syndrome (Ndufs4 knock-out (KO) mouse); however, the mechanism of rescue is unknown. Here we identify protein kinase C (PKC) downregulation as a key event mediating the beneficial effects of rapamycin treatment of Ndufs4 KO mice. Assessing the impact of rapamycin on the brain proteome and phosphoproteome of Ndufs4 KO mice, we find that rapamycin restores mitochondrial protein levels, inhibits signalling through both mTOR complexes and reduces the abundance and activity of multiple PKC isoforms. Administration of PKC inhibitors increases survival, delays neurological deficits, prevents hair loss and decreases inflammation in Ndufs4 KO mice. Thus, PKC may be a viable therapeutic target for treating severe mitochondrial disease.R01 NS098329 - NINDS NIH HHS; T32 HG00035 - U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI); T32 LM012419 - NLM NIH HHS; T32 HG000035 - NHGRI NIH HHS; R35 GM119536 - NIGMS NIH HHS; F32 NS110109 - NINDS NIH HHS; postdoctoral fellowship - Uehara Memorial Foundation; P30 AG013280 - NIA NIH HHS; postdoctoral fellowship - MEXT | Japan Society for the Promotion of Science (JSPS)Accepted manuscrip

Immunomagnetic microbeads for screening with flow cytometry and identification with nano-liquid chromatography mass spectrometry of ochratoxins in wheat and cereal

Multi-analyte binding assays for rapid screening of food contaminants require mass spectrometric identification of compound(s) in suspect samples. An optimal combination is obtained when the same bioreagents are used in both methods; moreover, miniaturisation is important because of the high costs of bioreagents. A concept is demonstrated using superparamagnetic microbeads coated with monoclonal antibodies (Mabs) in a novel direct inhibition flow cytometric immunoassay (FCIA) plus immunoaffinity isolation prior to identification by nano-liquid chromatography–quadrupole time-of-flight-mass spectrometry (nano-LC-Q-ToF-MS). As a model system, the mycotoxin ochratoxin A (OTA) and cross-reacting mycotoxin analogues were analysed in wheat and cereal samples, after a simple extraction, using the FCIA with anti-OTA Mabs. The limit of detection for OTA was 0.15 ng/g, which is far below the lowest maximum level of 3 ng/g established by the European Union. In the immunomagnetic isolation method, a 350-times-higher amount of beads was used to trap ochratoxins from sample extracts. Following a wash step, bound ochratoxins were dissociated from the Mabs using a small volume of acidified acetonitrile/water (2/8 v/v) prior to separation plus identification with nano-LC-Q-ToF-MS. In screened suspect naturally contaminated samples, OTA and its non-chlorinated analogue ochratoxin B were successfully identified by full scan accurate mass spectrometry as a proof of concept for identification of unknown but cross-reacting emerging mycotoxins. Due to the miniaturisation and bioaffinity isolation, this concept might be applicable for the use of other and more expensive bioreagents such as transport proteins and receptors for screening and identification of known and unknown (or masked) emerging food contaminants

Migrant remittances and the web of family obligations: Ongoing support among spatially extended kin in North-east Thailand, 1984–94

Exchanges of money, goods, and assistance among family/kin members are influenced by the intertwined lives of individuals and their family/kin. As people pass through the young adulthood years, acquiring obligations as spouses and parents, and migrating in search of economic opportunities, tensions can arise over existing obligations. Using rich longitudinal data from Northeast Thailand, we examined the role of family networks (origin and destination) on migrants’ exchanges with family/kin. Our approach overcame many shortcomings of earlier studies, allowing us to 'see' the family social network arrayed in a broader network. We show that intra-family exchanges are influenced by marital status, the presence of children, having parents in the origin household, and having siblings depart from it. The results are stable across sensitivity tests that systematically include or exclude various familial links. And reports provided by origin households on migrant remittances are consistent with reports from migrants themselves

Surveys of rice sold in Canada for aflatoxins, ochratoxin A and fumonisins

Approximately 200 samples of rice (including white, brown, red, black, basmati and jasmine, as well as wild rice) from several different countries, including the United States, Canada, Pakistan, India and Thailand, were analysed for aflatoxins, ochratoxin A (OTA) and fumonisins by separate liquid Chromatographic methods in two different years. The mean concentrations for aflatoxin B1 (AFB1) were 0.19 and 0.17 ng g−1 with respective positive incidences of 56% and 43% (≥ the limit of detection (LOD) of 0.002 ng g−1). Twenty-three samples analysed in the second year also contained aflatoxin B2 (AFB2) at levels ≥LOD of 0.002 ng g−1 The five most contaminated samples in each year contained 1.44–7.14 ng AFB1 g−1 (year 1) and 1.45–3.48 ng AFB1 g−1 (year 2); they were mostly basmati rice from India and Pakistan and black and red rice from Thailand. The average concentrations of ochratoxin A (OTA) were 0.05 and 0.005 ng g−1 in year 1 and year 2, respectively; incidences of samples containing ≥LOD of 0.05 ng g−1 were 43% and 1%, respectively, in the 2 years. All positive OTA results were confirmed by LC-MS/MS. For fumonisins, concentrations of fumonisin B1 (FB1) averaged 4.5 ng g−1 in 15 positive samples (≥0.7 ng g−1) from year 1 (n = 99); fumonisin B2 (FB2) and fumonisin B3 (FB3) were also present (≥1 ng g−1). In the second year there was only one positive sample (14 ng g−1 FB1) out of 100 analysed. All positive FB1 results were confirmed by LC-MS/MS

Predictors of nurturant parenting in teen mothers living in three generational families

Direct and indirect effects of grandparents on maternal nurturance in teen mothers (TM) living in three-generational families were explored with path analytic techniques in a sample of 107 working-class families. Perceived support from the teen's mother, grandparents' nurturance toward the baby, and the presence of the grandfather as a father figure in the home were hypothesized as increasing TM nurturance. TM nurturance was found to be positively predicted by grandparent nurturance and negatively predicted by TM perceived support from her mother. The strongest predictor of TM nurturance was grandfather nurturance toward the baby.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43953/1/10578_2006_Article_BF02353198.pd