How satellite cells and their progenitors balance differentiation and self-renewal to achieve sustainable tissue regeneration is not well understood. A major roadblock to understanding satellite cell fate decisions has been the difficulty to study this process in vivo . By visualizing expression dynamics of myogenic transcription factors during early regeneration in vivo , we identified the time point at which cells undergo decisions to differentiate or self-renew. Single-cell RNA sequencing revealed heterogeneity of satellite cells during both muscle homeostasis and regeneration, including a subpopulation enriched in Notch2 receptor expression. Furthermore, we reveal that differentiating cells express the Dll1 ligand. Using antagonistic antibodies we demonstrate that the DLL1 and NOTCH2 signaling pair is required for satellite cell self-renewal. Thus, differentiating cells provide the self-renewing signal during regeneration, enabling proportional regeneration in response to injury while maintaining the satellite cell pool. These findings have implications for therapeutic control of muscle regeneration.

The cellular origin of sporadic pancreatic neuroendocrine tumors (PNETs) is obscure. Hormone expression suggests that these tumors arise from glucagon-producing alpha cells or insulin-producing β cells, but instability in hormone expression prevents linage determination. We utilize loss of hepatic glucagon receptor (GCGR) signaling to drive alpha cell hyperproliferation and tumor formation to identify a cell of origin and dissect mechanisms that drive progression. Using a combination of genetically engineered Gcgr knockout mice and GCGR-inhibiting antibodies, we show that elevated plasma amino acids drive the appearance of a proliferative population of SLC38A5+ embryonic progenitor-like alpha cells in mice. Further, we characterize tumors from patients with rare bi-allelic germline GCGR loss-of-function variants and find prominent tumor-cell-associated expression of the SLC38A5 paralog SLC7A8 as well as markers of active mTOR signaling. Thus, progenitor cells arise from adult alpha cells in response to metabolic signals and, when inductive signals are chronically present, drive tumor initiation.

The cellular origin of sporadic pancreatic neuroendocrine tumors (PNETs) is obscure. Dysregulated hormone production and the absence of obvious driver mutations mask tumor origins although they presumably arise from endocrine cell lineages such as glucagon-producing alpha cells or insulin-expressing beta cells. A reciprocal feedback circuit involving pancreatic glucagon and hepatic amino acids regulates alpha cell mass such that loss of hepatic glucagon receptor (GCGR) signaling drives alpha cell hyperproliferation and tumor formation. This genetically-defined system is an ideal model for parsing the mechanisms of PNET origin and progression. Utilizing a combination of genetically engineered Gcgr knockout mice and GCGR-inhibiting antibodies, we found that elevated plasma amino acids drive reversion to a proliferative SLC38A5+ embryonic progenitor-like status. Chronic activation of this mTOR-dependent state drives tumor initiation and single cell profiling reveals tumors originate from a subpopulation of alpha cells. Profiling RNA, tumor signaling, and immune content of biopsies from rare human PNETs found in individuals with germ-line GCGR loss-of-function variants showed active mTOR and prominent tumor cell-associated expression of amino acid transporter SLC7A8. This work, for the first time, identifies metabolic control of an adult-to-progenitor alpha cell identity switch and shows that chronic maintenance of progenitor identity in the adult organ drives proliferation and tumor initiation.

In response to environmental and nutrient stress, adipose tissues must establish a new homeostatic state. Here we show that cold exposure of obese mice triggers an adaptive tissue remodeling in visceral adipose tissue (VAT) that involves extracellular matrix deposition, angiogenesis, sympathetic innervation, and adipose tissue browning. Obese VAT is predominated by pro-inflammatory M1 macrophages; cold exposure induces an M1-to-M2 shift in macrophage composition and dramatic changes in macrophage gene expression in both M1 and M2 macrophages. Antibody-mediated CSF1R blocking prevented the cold-induced recruitment of adipose tissue M2 macrophages, suggesting the role of CSF1R signaling in the process. These cold-induced effects in obese VAT are phenocopied by an administration of the FGF21-mimetic antibody, consistent with its action to stimulate sympathetic nerves. Collectively, these studies illuminate adaptive visceral adipose tissue plasticity in obese mice in response to cold stress and antibody-based metabolic therapy.

Objective: Fibroblast Growth Factor 21 (FGF21) is a potent stimulator of brown fat thermogenesis that improves insulin sensitivity, ameliorates hepatosteatosis, and induces weight loss by engaging the receptor complex comprised of Fibroblast Growth Factor Receptor 1 (FGFR1) and the requisite coreceptor beta Klotho. Previously, recombinant antibody proteins that activate the FGFR1/beta Klotho complex were proposed to act as an FGF21-mimetic; however, in vivo action of these engineered proteins has not been well studied. Methods: We investigated the mechanism by which anti-FGFR1/beta Klotho bispecific antibody (bFKB1) stimulates thermogenesis in UCP1-expressing brown adipocytes using genetically engineered mice. Anti-FGFR1 agonist antibody was also used to achieve brown adipose tissue restricted activation in transgenic mice. Results: Studies with global Ucp1-deficient mice and adipose-specific Fgfr1 deficient mice demonstrated that bFKB1 acts on targets distal to adipocytes and indirectly stimulates brown adipose thermogenesis in a UCP1-independent manner. Using a newly developed transgenic system, we also show that brown adipose tissue restricted activation of a transgenic FGFR1 expressed under the control of Ucp1 promoter does not stimulate energy expenditure. Finally, consistent with its action as a FGF21 mimetic, bFBK1 suppresses intake of saccharin-containing food and alcohol containing water in mice. Conclusions: Collectively, we propose that FGFR1/beta Klotho targeted therapy indeed mimics the action of FGF21 in vivo and stimulates UCP1-independent brown fat thermogenesis through receptors outside of adipocytes and likely in the nervous system. (C) 2017 Genentech, Inc. Published by Elsevier GmbH.

Understanding the regulation of islet cell mass has important implications for the discovery of regenerative therapies for diabetes. The liver plays a central role in metabolism and the regulation of endocrine cell number, but liver-derived factors that regulate α-cell and β-cell mass remain unidentified. We propose a nutrient-sensing circuit between liver and pancreas in which glucagon-dependent control of hepatic amino acid metabolism regulates α-cell mass. We found that glucagon receptor inhibition reduced hepatic amino acid catabolism, increased serum amino acids, and induced α-cell proliferation in an mTOR-dependent manner. In addition, mTOR inhibition blocked amino-acid-dependent α-cell replication ex vivo and enabled conversion of α-cells into β-like cells in vivo. Serum amino acids and α-cell proliferation were increased in neonatal mice but fell throughout postnatal development in a glucagon-dependent manner. These data reveal that amino acids act as sensors of glucagon signaling and can function as growth factors that increase α-cell proliferation.

Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT) has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR) 1/βKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/βKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/βKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/βKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/βKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin.

In pre-clinical mouse models, the interleukin-22 pathway is identified as a novel target for therapeutic intervention in metabolic diseases.

Background: Allosteric regulators of GPCRs provide unique pharmacological properties. Results: The mechanism of allosteric inhibition of the glucagon receptor by an antibody, which is uniquely sensitive to a naturally occurring G40S mutation, is detailed. Conclusion: Allosteric sites on the glucagon receptor extracellular domain regulate receptor activity. Significance: Mechanisms of allosteric regulation of GPCRs aid discovery of drugs with improved selectivity. Elevated glucagon levels and increased hepatic glucagon receptor (GCGR) signaling contribute to hyperglycemia in type 2 diabetes. We have identified a monoclonal antibody that inhibits GCGR, a class B G-protein coupled receptor (GPCR), through a unique allosteric mechanism. Receptor inhibition is mediated by the binding of this antibody to two distinct sites that lie outside of the glucagon binding cleft. One site consists of a patch of residues that are surface-exposed on the face of the extracellular domain (ECD) opposite the ligand-binding cleft, whereas the second binding site consists of residues in the αA helix of the ECD. A docking model suggests that the antibody does not occlude the ligand-binding cleft. We solved the crystal structure of GCGR ECD containing a naturally occurring G40S mutation and found a shift in the register of the αA helix that prevents antibody binding. We also found that alterations in the αA helix impact the normal function of GCGR. We present a model for the allosteric inhibition of GCGR by a monoclonal antibody that may form the basis for the development of allosteric modulators for the treatment of diabetes and other class B GPCR-related diseases.

Antibody-mediated activation of fibroblast growth factor receptor 1 reverses the diabetic phenotype in mice, likely by affecting brown adipose tissues.