David W. Scheel

Human BAT Possesses Molecular Signatures That Resemble Beige/Brite Cells

Brown adipose tissue (BAT) dissipates chemical energy and generates heat to protect animals from cold and obesity. Rodents possess two types of UCP-1 positive brown adipocytes arising from distinct developmental lineages: “classical” brown adipocytes develop during the prenatal stage whereas “beige” or “brite” cells that reside in white adipose tissue (WAT) develop during the postnatal stage in response to chronic cold or PPARγ agonists. Beige cells’ inducible characteristics make them a promising therapeutic target for obesity treatment, however, the relevance of this cell type in humans remains unknown. In the present study, we determined the gene signatures that were unique to classical brown adipocytes and to beige cells induced by a specific PPARγ agonist rosiglitazone in mice. Subsequently we applied the transcriptional data to humans and examined the molecular signatures of human BAT isolated from multiple adipose depots. To our surprise, nearly all the human BAT abundantly expressed beige cell-selective genes, but the expression of classical brown fat-selective genes were nearly undetectable. Interestingly, expression of known brown fat-selective genes such as PRDM16 was strongly correlated with that of the newly identified beige cell-selective genes, but not with that of classical brown fat-selective genes. Furthermore, histological analyses showed that a new beige cell marker, CITED1, was selectively expressed in the UCP1-positive beige cells as well as in human BAT. These data indicate that human BAT may be primary composed of beige/brite cells.

Gene expression cascades in pancreatic development

The specialized endocrine and exocrine cells of the pancreas originally derive from a pool of apparently identical cells in the early gut endoderm. Serial changes in their gene expression program, controlled by a hierarchy of pancreatic transcription factors, direct this progression from multipotent progenitor cell to mature pancreatic cell. When the cells differentiate, this hierarchy of factors coalesces into a network of factors that maintain the differentiated phenotype of the cells. As we develop an understanding of the pancreatic transcription factors, we are also acquiring the tools with which we can ultimately control pancreatic cell differentiation.

Rfx6 directs islet formation and insulin production in mice and humans

Insulin from the β-cells of the pancreatic islets of Langerhans controls energy homeostasis in vertebrates, and its deficiency causes diabetes mellitus. During embryonic development, the transcription factor neurogenin 3 (Neurog3) initiates the differentiation of the β-cells and other islet cell types from pancreatic endoderm, but the genetic program that subsequently completes this differentiation remains incompletely understood. Here we show that the transcription factor Rfx6 directs islet cell differentiation downstream of Neurog3. Mice lacking Rfx6 failed to generate any of the normal islet cell types except for pancreatic-polypeptide-producing cells. In human infants with a similar autosomal recessive syndrome of neonatal diabetes, genetic mapping and subsequent sequencing identified mutations in the human RFX6 gene. These studies demonstrate a unique position for Rfx6 in the hierarchy of factors that coordinate pancreatic islet development in both mice and humans. Rfx6 could prove useful in efforts to generate β-cells for patients with diabetes.

Transient cytokine treatment induces acinar cell reprogramming and regenerates functional beta cell mass in diabetic mice

Reprogramming of pancreatic exocrine cells into cells resembling beta cells may provide a strategy for treating diabetes. Here we show that transient administration of epidermal growth factor and ciliary neurotrophic factor to adult mice with chronic hyperglycemia efficiently stimulates the conversion of terminally differentiated acinar cells to beta-like cells. Newly generated beta-like cells are epigenetically reprogrammed, functional and glucose responsive, and they reinstate normal glycemic control for up to 248 d. The regenerative process depends on Stat3 signaling and requires a threshold number of Neurogenin 3 (Ngn3)-expressing acinar cells. In contrast to previous work demonstrating in vivo conversion of acinar cells to beta-like cells by viral delivery of exogenous transcription factors, our approach achieves acinar-to-beta-cell reprogramming through transient cytokine exposure rather than genetic modification.

Autoregulation and Maturity Onset Diabetes of the Young Transcription Factors Control the Human PAX4 Promoter

During pancreatic development, the paired homeodomain transcription factor PAX4 is required for the differentiation of the insulin-producing beta cells and somatostatin-producing delta cells. To establish the position of PAX4 in the hierarchy of factors controlling islet cell development, we examined the control of the human PAX4 gene promoter. In both cell lines and transgenic animals, a 4.9-kilobase pair region directly upstream of the human PAX4 gene transcriptional start site acts as a potent pancreas-specific promoter. Deletion mapping experiments demonstrate that a 118-base pair region lying approximately 1.9 kilobase pairs upstream of the transcription start site is both necessary and sufficient to direct pancreas-specific expression. Serial deletions through this region reveal the presence of positive elements that bind several pancreatic transcription factors as follows: the POU homeodomain factor HNF1α, the orphan nuclear receptor HNF4α, the homeodomain factor PDX1, and a heterodimer composed of two basic helix-loop-helix factors. Interestingly, mutations in the genes encoding four of these factors cause a dominantly inherited form of human diabetes called Maturity Onset Diabetes of the Young. In addition, PAX4 itself has at least two high affinity binding sites within the promoter through which it exerts a strong negative autoregulatory effect. Together, these results suggest a model in which PAX4 expression is activated during pancreatic development by a combination of pancreas-specific factors but is then switched off once PAX4 protein reaches sufficient levels.

Menin determines K-RAS proliferative outputs in endocrine cells.

Endocrine cell proliferation fluctuates dramatically in response to signals that communicate hormone demand. The genetic alterations that override these controls in endocrine tumors often are not associated with oncogenes common to other tumor types, suggesting that unique pathways govern endocrine proliferation. Within the pancreas, for example, activating mutations of the prototypical oncogene KRAS drive proliferation in all pancreatic ductal adenocarcimomas but are never found in pancreatic endocrine tumors. Therefore, we asked how cellular context impacts K-RAS signaling. We found that K-RAS paradoxically suppressed, rather than promoted, growth in pancreatic endocrine cells. Inhibition of proliferation by K-RAS depended on antiproliferative RAS effector RASSF1A and blockade of the RAS-activated proproliferative RAF/MAPK pathway by tumor suppressor menin. Consistent with this model, a glucagon-like peptide 1 (GLP1) agonist, which stimulates ERK1/2 phosphorylation, did not affect endocrine cell proliferation by itself, but synergistically enhanced proliferation when combined with a menin inhibitor. In contrast, inhibition of MAPK signaling created a synthetic lethal interaction in the setting of menin loss. These insights suggest potential strategies both for regenerating pancreatic β cells for people with diabetes and for targeting menin-sensitive endocrine tumors.

Phosphoproteomics Identifies CK2 as a Negative Regulator of Beige Adipocyte Thermogenesis and Energy Expenditure

Catecholamines promote lipolysis both in brown and white adipocytes, whereas the same stimuli preferentially activate thermogenesis in brown adipocytes. Molecular mechanisms for the adipose-selective activation of thermogenesis remain poorly understood. Here, we employed quantitative phosphoproteomics to map global and temporal phosphorylation profiles in brown, beige, and white adipocytes under β3-adrenenoceptor activation and identified kinases responsible for the adipose-selective phosphorylation profiles. We found that casein kinase2 (CK2) activity is preferentially higher in white adipocytes than brown/beige adipocytes. Genetic or pharmacological blockade of CK2 in white adipocytes activates the thermogenic program in response to cAMP stimuli. Such activation is largely through reduced CK2-mediated phosphorylation of class I HDACs. Notably, inhibition of CK2 promotes beige adipocyte biogenesis and leads to an increase in whole-body energy expenditure and ameliorates diet-induced obesity and insulin resistance. These results indicate that CK2 is a plausible target to rewire the β3-adrenenoceptor signaling cascade that promotes thermogenesis in adipocytes.

Gαi/o-coupled receptor signaling restricts pancreatic β-cell expansion

Significance This paper shows that a class of receptors known to modulate insulin release by pancreatic β cells also regulates the proliferation of these cells and restrains the perinatal β-cell expansion that establishes adult β-cell mass, suggesting that alterations in signaling by these receptors could contribute to the decreased β-cell numbers seen in patients with type 2 diabetes. Further, inhibition of signaling through these receptors potentially could be used to generate more β cells for people with diabetes. Gi-GPCRs, G protein-coupled receptors that signal via Gα proteins of the i/o class (Gαi/o), acutely regulate cellular behaviors widely in mammalian tissues, but their impact on the development and growth of these tissues is less clear. For example, Gi-GPCRs acutely regulate insulin release from pancreatic β cells, and variants in genes encoding several Gi-GPCRs—including the α-2a adrenergic receptor, ADRA2A—increase the risk of type 2 diabetes mellitus. However, type 2 diabetes also is associated with reduced total β-cell mass, and the role of Gi-GPCRs in establishing β-cell mass is unknown. Therefore, we asked whether Gi-GPCR signaling regulates β-cell mass. Here we show that Gi-GPCRs limit the proliferation of the insulin-producing pancreatic β cells and especially their expansion during the critical perinatal period. Increased Gi-GPCR activity in perinatal β cells decreased β-cell proliferation, reduced adult β-cell mass, and impaired glucose homeostasis. In contrast, Gi-GPCR inhibition enhanced perinatal β-cell proliferation, increased adult β-cell mass, and improved glucose homeostasis. Transcriptome analysis detected the expression of multiple Gi-GPCRs in developing and adult β cells, and gene-deletion experiments identified ADRA2A as a key Gi-GPCR regulator of β-cell replication. These studies link Gi-GPCR signaling to β-cell mass and diabetes risk and identify it as a potential target for therapies to protect and increase β-cell mass in patients with diabetes.

A mouse model for monitoring islet cell genesis and developing therapies for diabetes.

SUMMARY Transient expression of the transcription factor neurogenin-3 marks progenitor cells in the pancreas as they differentiate into islet cells. We developed a transgenic mouse line in which the surrogate markers secreted alkaline phosphatase (SeAP) and enhanced green florescent protein (EGFP) can be used to monitor neurogenin-3 expression, and thus islet cell genesis. In transgenic embryos, cells expressing EGFP lined the pancreatic ducts. SeAP was readily detectable in embryos, in the media of cultured embryonic pancreases and in the serum of adult animals. Treatment with the γ-secretase inhibitor DAPT, which blocks Notch signaling, enhanced SeAP secretion rates and increased the number of EGFP-expressing cells as assayed by fluorescence-activated cell sorting (FACS) and immunohistochemistry in cultured pancreases from embryos at embryonic day 11.5, but not in pancreases harvested 1 day later. By contrast, treatment with growth differentiation factor 11 (GDF11) reduced SeAP secretion rates. In adult mice, partial pancreatectomy decreased, whereas duct ligation increased, circulating SeAP levels. This model will be useful for studying signals involved in islet cell genesis in vivo and developing therapies that induce this process.