SUMMARY Increasing energy expenditure through activation of endogenous brown adipose tissue (BAT) is a potential approach to treat obesity and diabetes. The class of β3-adrenergic receptor (AR) agonists stimulates rodent BAT, but this activity has never been demonstrated in humans. Here we determined the ability of 200 mg oral mirabegron (Myrbetriq, Astellas Pharma, Inc.), a β3-AR agonist currently approved to treat overactive bladder, to stimulate BAT as compared to placebo. Mirabegron led to higher BAT metabolic activity as measured via 18F-fluorodeoxyglucose (18F-FDG) using positron emission tomography (PET) combined with computed tomography (CT) in all twelve healthy male subjects (p = 0.001), and it increased resting metabolic rate (RMR) by 203 ± 40 kcal/day (+13%; p = 0.001). BAT metabolic activity was also a significant predictor of the changes in RMR (p = 0.006). Therefore, a β3-AR agonist can stimulate human BAT thermogenesis and may be a promising treatment for metabolic disease.

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Filamentous phage infection induces the synthesis of large amounts of an Escherichia coli protein, phage shock protein (Psp), the product of a previously undescribed gene. This induction is due to the phage gene IV protein, pIV, an integral membrane protein. The uninduced level of Psp is undetectable, but when induced by prolonged synthesis of pIV, it can become one of the most abundant proteins in the cell. Psp is also synthesized transiently in response to several stresses (heat, ethanol, and osmotic shock). High-level synthesis occurs only after extreme treatment. Unlike the members of the heat shock regulon, Psp induction does not require the heat shock sigma factor, sigma 32; some stimuli that elicit sigma 32-dependent heat shock proteins do not induce Psp synthesis. The level of Psp induction after extreme stress is even higher in sigma 32 mutant cells, which are unable to mount a normal heat shock response, suggesting that these parallel stress responses are interrelated.

Targeting brown adipose tissue (BAT) content or activity has therapeutic potential for treating obesity and the metabolic syndrome by increasing energy expenditure. Both inter- and intra-individual differences contribute to heterogeneity in human BAT and potentially to differential thermogenic capacity in human populations. Here, we demonstrated the generated clones of brown and white preadipocytes from human neck fat of four individuals and characterized their adipogenic differentiation and thermogenic function. Combining an uncoupling protein 1(UCP1) reporter system and expression profiling, we defined novel sets of gene signatures in human preadipocytes that could predict the thermogenic potential of the cells once they were maturated in culture. Knocking out the positive UCP1 regulators identified by this approach, PREX1 and EDNRB in brown preadipocytes using CRISPR/Cas9 markedly abolished the high level of UCP1 in brown adipocytes differentiated from the preadipocytes. Finally, we were able to prospectively isolate adipose progenitors with great thermogenic potential using cell surface marker CD29. These data provide new insights into the cellular heterogeneity in human fat and offer the identification of possible biomarkers of thermogenically competent preadipocytes.

The recent discovery of the human coun-terpart of the hairlessmousephenotype1has helped our understandingof the molecular genetics of hair growth.But there are no reports of a defect in thehuman homologue of the best known of the'bald' mouse phenotypes, the nudemouse2.This may be because affected individualsare so gravely ill from the accompanyingimmunodeficiency that their baldness goesunnoticed. We have carried out a geneticanalysis that reveals a human homologue ofthe nudemouse.The nudemouse is characterized by acongenital absence of hair and a severeimmunodeficiency2, resulting from muta-tions in the whn(winged-helix-nude;Hfh11nu) gene, which encodes a member ofthe forkhead/winged-helix transcriptionfactor family with restricted expression inthymus and skin3. The simultaneous occur-rence of severe functional T-cell immunodeficiency, congenital alopecia and nail dys-trophy (MIM database no. 601705) in twoaffected sisters led to the recognition thatthe clinical phenotype was reminiscent ofthe nudemouse4. We therefore investigatedwhether this syndrome represents thehuman counterpart of the nudemousephenotype.We obtained DNA samples from mem-bers of the sisters' family in a small villagein southern Italy. The affected sisters wereborn with a complete absence of scalp hair (Fig. 1a), eyebrows and eyelashes and haddystrophic nails, and no thymic shadow wasevident upon X-ray examination. The firstaffected child revealed a striking impair-ment of T-cell function shortly after birth,and died at the age of 12 months. Her sisterhad similar immunological abnormalities,but bonemarrow transplantation at fivemonths of age led to full immunologicalreconstitution, although the alopecia andnail dystrophy are still present4.We performed linkage analysis usingmicrosatellite markers near the humanWHNlocus on chromosome 17, and founda lod score of 1.32, suggestive of linkage. Wethen sequenced the human WHNgene5andfound a homozygous C-to-T transition atnucleotide position 792 of the WHNcDNA(GenBank accession no. Y11739) (Fig. 1b).This leads to a nonsense mutation atresidue 255 (R255X) in exon 5, and predictsthe complete absence of functional proteinas a result of nonsense-mediated decay ofmessenger RNA.Because the proband's bonemarrowtransplant was from her brother, we exam-ined her leukocyte DNA both before andafter the graft for the presence of chi-maerism. Genotyping the proband beforethe transplant showed that her leukocyteDNA was homozygous only for the mutantallele (Fig. 1c). Four years after the transplant, we detected the haplotype specific forthe wild-type paternal WHNallele receivedfrom the brother, as well as the mutantallele, indicative of chimaerism. Genderdetermination revealed that the proband'sleukocyte DNA was genotypically XXbefore the transplant, and the brother'sDNA was XY. Afterwards, the proband'sleukocyte DNA was found to be XY (Fig.1c), providing evidence of longtermengraftment and expansion of the bone-marrow graft.The WHNgene encodes a transcriptionfactor, which is developmentally regulatedand directs cel...

The phage shock protein (psp) operon (pspABCE) of Escherichia coli is strongly induced in response to a variety of stressful conditions or agents such as filamentous phage infection, ethanol treatment, osmotic shock, heat shock, and prolonged incubation in stationary phase. Transcription of the psp operon is driven from a The phage shock protein (psp) operon of Escherichia coli is specifically and continually induced by gene IV protein (pIV) of filamentous phages (8, 47). Psp genes are also synthesized transiently in response to several stresses such as heat shock, osmotic shock, and ethanol treatment (8). Inhibition of protein secretion or lipid biosynthesis, treatment with ionophores or free fatty acids, and prolonged stationary-phase incubation induce the psp operon as well (5,11,28,59). Heat shock, osmotic shock, and ethanol treatment also induce the heat shock response in E. coli (33,60). Although the psp operon is induced by many stimuli that also elicit the heat shock response, psp expression does not require the heat shock sigma factor, 32 (9). Indeed, in 32 mutants, psp expression is elevated during unstressed growth, and the response to heat or ethanol shock is prolonged (8,9,56). The function of the psp operon in E. coli physiology is not clear, but bacteria that lack the psp operon are less able to survive prolonged incubation in stationary phase at pH 9.0, show increased motility (58, 59), and exhibit a reduction in the efficiency of protein translocation (27a, 28).The psp operon consists of four genes, pspABCE, and its expression is controlled principally at the transcriptional level (9,28,57). All psp transcription is driven by the alternative holoenzyme form of RNA polymerase (RNAP) containing the 54 factor ( 54

The phage shock protein (psp) operon of Escherichia coli is strongly induced in response to heat, ethanol, osmotic shock, and infection by filamentous bacteriophages. The operon contains at least four genes--pspA, pspB, pspC, and pspE--and is regulated at the transcriptional level. We report here that psp expression is controlled by a network of positive and negative regulatory factors and that transcription in response to all inducing agents is directed by the or-factor r s4. Negative regulation is mediated by both PspA and the r heat shock proteins. The PspB and PspC proteins cooperatively activate expression, possibly by antagonizing the PspA-controlled repression. The strength of this activation is determined primarily by the concentration of PspC, whereas PspB enhances but is not absolutely essential for PspC-dependent expression. PspC is predicted to contain a leucine zipper, a motif responsible for the dimerization of many eukaryotic transcriptional activators. PspB and PspC, though not necessary for psp expression during heat shock, are required for the strong psp response to phage infection, osmotic shock, and ethanol treatment. The psp operon thus represents a third category of transcriptional control mechanisms, in addition to the r 32-and erE-dependent systems, for genes induced by heat and other stresses.[Key Words: Phage shock protein; stress response; heat shock; cr54; filamentous bacteriophage; leucine zipper] Received June 20, 1991; revised version accepted August 15, 1991.Exposure to certain adverse environmental conditions, such as high temperature, causes all organisms to coordinately and vigorously induce the synthesis of a specific set of proteins called the heat shock proteins (HSPs; for reviews, see Lindquist and Craig 1988;Gross et al. 1990). This phenomenon, the heat shock response, is the product of perhaps the best conserved and most universal genetic network. Similarities in this response between prokaryotes and eukaryotes include the sequences of certain HSPs (e.g., the 90-, 70-, and 60-kD HSP families), the treatments that stimulate the response (e.g., heat, ethanol, heavy metal ions), and the large, rapid increases in heat shock gene transcription that follow environmental challenge. In Escherichia coli, previous work in several laboratories identified at least two mechanisms of transcriptional control for heat shock gene expression. Most of the detected heat shock genes (-17)

The phage shock protein operon (pspABCE) ofEscherichia coli is strongly expressed in response to stress environmental conditions, such as heat shock, ethanol treatment, osmotic shock, and filamentous phage infection. We show that bacteria lacking the pspABC genes exhibit a substantial decrease in the ability to survive prolonged incubation in stationary phase under alkaline conditions (pH 9). The psp mutant bacteria grow approximately as well as wild-type strains in the alkaline medium, and stationary-phase survival of the psp mutants improves substantially at pH values closer to the optimal growth range (pH 6-8). In late statoar-phase (1-to 2-day-old) cells, the operon can be strongly induced under certain conditions, and PspA can become one ofthe most highly expressed bacterial proteins. The combination of stationary-phase starvation and alkaline pH is likely to place a severe strain on the maintenance ofendogenous energy sources, and, consistent with these effects, we find that psp expression is also induced by uncouplers of oxidative phosphorylation and other agents that interfere with energy production. (1)], and the existence of a multifactor network tightly regulating psp expression, led us to assume that the operon serves a protective or beneficial function. We have suggested that the lack of a stress-related, logarithmic-phase psp phenotype is due to a functional overlap ofthe Psps with the cr32-controlled regulon (6). Both PspA and the o-32-dependent heat shock system turn off psp expression, and this negative regulation may result from similar or overlapping activities.We report here that the psp operon is required for prolonged survival in stationary phase at alkaline pH (pH 9) and that the expression of the psp genes can be strongly induced during stationary phase under certain conditions. MATERIALS AND METHODSBacterial Strains and Plasmids. Strains K561, J134 (K561 ApspABC), L1, and L24 (L1 ApspABC) have been described (6, 10). SG20045 (cps-5::TnlO Alon-100) (11) was kindly provided by Susan Gottesman (National Institutes of Health). SK5022 (zci-604: :TnlO) was from the E. coli Genetic Stock Center (CGSC no. 6666). L104 (K561 cps-S::TnlO), L102 (J134 cps-S::TnJO), and L96 (J134 psp+ zci-604:: TnlO) were generated by transduction (12). The plasmid pBRPS-1, which carries the complete psp operon, was constructed by ligating the 4.5-kb EcoRI fragment of pPS-1 (5)