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1.
bioRxiv ; 2024 May 03.
Article in English | MEDLINE | ID: mdl-38746433

ABSTRACT

Islet transplantation can cure type 1 diabetes, but peri-transplant beta cell death limits this procedure to those with low insulin requirements. Improving human beta cell survival or proliferation may make islet transplantation a possibility for more type 1 patients. To identify novel regulators of beta cell survival and proliferation, we conducted a pooled small hairpin RNA (shRNA) screen in primary human beta cells transplanted into immunocompromised mice. shRNAs targeting several cyclin dependent kinase inhibitors were enriched after transplant. Here, we focused on the Gi/o-coupled GPCR, serotonin 1F receptor ( HTR1F, 5-HT 1F ) which our screen identified as a negative regulator of beta cell numbers after transplant. In vitro , 5-HT 1F knockdown induced human beta cell proliferation but only when combined with harmine and exendin-4. In vivo , knockdown of 5-HT 1F reduced beta cell death during transplant. To demonstrate the feasibility of targeting 5-HT 1F in islet transplant, we identified and validated a small molecule 5-HT 1F antagonist. This antagonist increased glucose stimulated insulin secretion from primary human islets and cAMP accumulation in primary human beta cells. Finally, the 5-HT 1F antagonist improved glycemia in marginal mass, human islet transplants into immunocompromised mice. We identify 5-HT 1F as a novel druggable target to improve human beta cell survival in the setting of islet transplantation. One Sentence Summary: Serotonin 1F receptor (5-HT 1F ) negatively regulates insulin secretion and beta cell survival during transplant.

2.
Cell Metab ; 33(7): 1322-1341.e13, 2021 07 06.
Article in English | MEDLINE | ID: mdl-34019840

ABSTRACT

Mitochondria control eukaryotic cell fate by producing the energy needed to support life and the signals required to execute programed cell death. The biochemical milieu is known to affect mitochondrial function and contribute to the dysfunctional mitochondrial phenotypes implicated in cancer and the morbidities of aging. However, the physical characteristics of the extracellular matrix are also altered in cancerous and aging tissues. Here, we demonstrate that cells sense the physical properties of the extracellular matrix and activate a mitochondrial stress response that adaptively tunes mitochondrial function via solute carrier family 9 member A1-dependent ion exchange and heat shock factor 1-dependent transcription. Overall, our data indicate that adhesion-mediated mechanosignaling may play an unappreciated role in the altered mitochondrial functions observed in aging and cancer.


Subject(s)
Cell Adhesion/physiology , Mechanotransduction, Cellular/physiology , Mitochondrial Dynamics/physiology , Adult , Animals , Animals, Genetically Modified , Caenorhabditis elegans , Cell Respiration , Cells, Cultured , Extracellular Matrix/metabolism , Female , HEK293 Cells , Humans , Hyperglycemia/metabolism , Hyperglycemia/pathology , Hyperglycemia/physiopathology , Integrins/physiology , Ion Exchange , Mice , Microscopy, Confocal , Middle Aged , Mitochondria/metabolism , Mitochondria/physiology , Oxidative Stress/physiology , Reactive Oxygen Species/metabolism , Signal Transduction/physiology , Sodium-Hydrogen Exchanger 1/physiology , Time-Lapse Imaging
3.
Sci Rep ; 10(1): 5629, 2020 03 27.
Article in English | MEDLINE | ID: mdl-32221326

ABSTRACT

Gpr27 is a highly conserved, orphan G protein coupled receptor (GPCR) previously implicated in pancreatic beta cell insulin transcription and glucose-stimulated insulin secretion in vitro. Here, we characterize a whole-body mouse knockout of Gpr27. Gpr27 knockout mice were born at expected Mendelian ratios and exhibited no gross abnormalities. Insulin and Pdx1 mRNA in Gpr27 knockout islets were reduced by 30%, but this did not translate to a reduction in islet insulin content or beta cell mass. Gpr27 knockout mice exhibited slightly worsened glucose tolerance with lower plasma insulin levels while maintaining similar insulin tolerance. Unexpectedly, Gpr27 deletion reduced expression of Eif4e3, a neighboring gene, likely by deleting transcription start sites on the anti-sense strand of the Gpr27 coding exon. Our data confirm that loss of Gpr27 reduces insulin mRNA in vivo but has only minor effects on glucose tolerance.


Subject(s)
Diabetes Mellitus/metabolism , Insulin/metabolism , RNA, Messenger/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , Glucose/metabolism , Insulin Secretion/physiology , Insulin-Secreting Cells , Islets of Langerhans/metabolism , Male , Mice , Mice, Knockout
4.
Endocrinology ; 159(9): 3321-3330, 2018 09 01.
Article in English | MEDLINE | ID: mdl-30059978

ABSTRACT

Insulin production by the pancreatic ß cell is critical for the glucose homeostasis of the whole organism. Although the transcription factors required for insulin production are known, the upstream pathways that control insulin production are less clear. To further elucidate this regulatory network, we created a genetic interaction map of insulin production by performing ∼20,000 pairwise RNA interference knockdowns of insulin promoter regulators. Our map correctly predicted known physical complexes in the electron transport chain and a role for Spry2 in the unfolded protein response. To further validate our map, we used it to predict the function of an unannotated gene encoding a 37-kDa protein with no identifiable domains we have termed mitochondrial fission factor interactor (Mfi). We have shown that Mfi is a binding partner of the mitochondrial fission factor and that Mfi inhibits dynamin-like protein 1 recruitment to mitochondria. Our data provide a resource to understand the regulatory network of insulin promoter activity.


Subject(s)
Insulin-Secreting Cells/metabolism , Insulin/biosynthesis , Mitochondria/metabolism , Mitochondrial Dynamics/genetics , Animals , Cell Line , Dynamins , GTP Phosphohydrolases , Gene Regulatory Networks , Humans , Insulin/genetics , Intracellular Signaling Peptides and Proteins , Membrane Proteins/metabolism , Mice , Microtubule-Associated Proteins , Mitochondrial Proteins/metabolism , Promoter Regions, Genetic/genetics , Unfolded Protein Response
5.
Endocrinology ; 159(9): 3245-3256, 2018 09 01.
Article in English | MEDLINE | ID: mdl-30052866

ABSTRACT

Mitochondria are dynamic organelles that undergo frequent fission and fusion events. Mitochondrial fission is required for ATP production, the tricarboxylic acid cycle, and processes beyond metabolism in a cell-type specific manner. Ex vivo and cell line studies have demonstrated that Drp1, a central regulator of mitochondrial fission, is required for glucose-stimulated insulin secretion (GSIS) in pancreatic ß cells. Herein, we set out to interrogate the role of Drp1 in ß-cell insulin secretion in vivo. We generated ß-cell-specific Drp1 knockout (KO) mice (Drp1ß-KO) by crossing a conditional allele of Drp1 to Ins1cre mice, in which Cre recombinase replaces the coding region of the Ins1 gene. Drp1ß-KO mice were glucose intolerant due to impaired GSIS but did not progress to fasting hyperglycemia as adults. Despite markedly abnormal mitochondrial morphology, Drp1ß-KO islets exhibited normal oxygen consumption rates and an unchanged glucose threshold for intracellular calcium mobilization. Instead, the most profound consequences of ß-cell Drp1 deletion were impaired second-phase insulin secretion and impaired glucose-stimulated amplification of insulin secretion. Our data establish Drp1 as an important regulator of insulin secretion in vivo and demonstrate a role for Drp1 in metabolic amplification and calcium handling without affecting oxygen consumption.


Subject(s)
Dynamins/genetics , Insulin Secretion/genetics , Insulin-Secreting Cells/metabolism , Mitochondria/metabolism , Oxygen Consumption/genetics , Animals , Calcium/metabolism , Fasting/metabolism , Glucose Intolerance/genetics , Hyperglycemia/genetics , Islets of Langerhans/metabolism , Mice , Mice, Knockout , Mitochondria/pathology , Mitochondrial Dynamics
6.
Diabetes ; 66(6): 1703-1712, 2017 06.
Article in English | MEDLINE | ID: mdl-28246293

ABSTRACT

Insulin production by the pancreatic ß-cell is required for normal glucose homeostasis. While key transcription factors that bind to the insulin promoter are known, relatively little is known about the upstream regulators of insulin transcription. Using a whole-genome RNA interference screen, we uncovered 26 novel regulators of insulin transcription that regulate diverse processes including oxidative phosphorylation, vesicle traffic, and the unfolded protein response (UPR). We focused on Spry2-a gene implicated in human type 2 diabetes by genome-wide association studies but without a clear connection to glucose homeostasis. We showed that Spry2 is a novel UPR target and its upregulation is dependent on PERK. Knockdown of Spry2 resulted in reduced expression of Serca2, reduced endoplasmic reticulum calcium levels, and induction of the UPR. Spry2 deletion in the adult mouse ß-cell caused hyperglycemia and hypoinsulinemia. Our study greatly expands the compendium of insulin promoter regulators and demonstrates a novel ß-cell link between Spry2 and human diabetes.


Subject(s)
Diabetes Mellitus, Type 2/genetics , Gene Expression Regulation/genetics , Insulin-Secreting Cells/metabolism , Insulin/genetics , Intracellular Signaling Peptides and Proteins/genetics , Membrane Proteins/genetics , Unfolded Protein Response/genetics , Animals , Annexin A5/metabolism , Blotting, Western , Calcium/metabolism , Cell Line , Diabetes Mellitus, Type 2/metabolism , Endoplasmic Reticulum/metabolism , Gene Knockdown Techniques , Genome-Wide Association Study , Humans , Hyperglycemia/genetics , Hyperglycemia/metabolism , Insulin/metabolism , Mice , Protein Serine-Threonine Kinases , RNA Interference , Real-Time Polymerase Chain Reaction , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , eIF-2 Kinase/metabolism
7.
J Clin Invest ; 124(9): 4093-101, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25133424

ABSTRACT

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 ß cells for people with diabetes and for targeting menin-sensitive endocrine tumors.


Subject(s)
Islets of Langerhans/cytology , Proto-Oncogene Proteins/physiology , ras Proteins/physiology , Adult , Animals , Cell Proliferation , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Humans , Male , Mice , Middle Aged , Phosphorylation , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins p21(ras) , Signal Transduction , Tumor Suppressor Proteins/physiology
8.
Mol Endocrinol ; 26(10): 1783-92, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22915829

ABSTRACT

The pancreatic ß-cell is critical for the maintenance of glycemic control. Knowing the compendium of genes expressed in ß-cells will further our understanding of this critical cell type and may allow the identification of future antidiabetes drug targets. Here, we report the use of next-generation sequencing to obtain nearly 1 billion reads from the polyadenylated RNA of islets and purified ß-cells from mice. These data reveal novel examples of ß-cell-specific splicing events, promoter usage, and over 1000 long intergenic noncoding RNA expressed in mouse ß-cells. Many of these long intergenic noncoding RNA are ß-cell specific, and we hypothesize that this large set of novel RNA may play important roles in ß-cell function. Our data demonstrate unique features of the ß-cell transcriptome.


Subject(s)
High-Throughput Nucleotide Sequencing , Insulin-Secreting Cells/metabolism , Transcriptome , Animals , Female , Gene Expression Profiling , Male , Mice , Mice, Transgenic , Organ Specificity , Promoter Regions, Genetic , RNA Splicing , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Analysis, RNA , Tryptophan Hydroxylase/genetics , Tryptophan Hydroxylase/metabolism
9.
PLoS Genet ; 8(1): e1002449, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22253604

ABSTRACT

The prevalence of type 2 diabetes in the United States is projected to double or triple by 2050. We reasoned that the genes that modulate insulin production might be new targets for diabetes therapeutics. Therefore, we developed an siRNA screening system to identify genes important for the activity of the insulin promoter in beta cells. We created a subclone of the MIN6 mouse pancreatic beta cell line that expresses destabilized GFP under the control of a 362 base pair fragment of the human insulin promoter and the mCherry red fluorescent protein under the control of the constitutively active rous sarcoma virus promoter. The ratio of the GFP to mCherry fluorescence of a cell indicates its insulin promoter activity. As G protein coupled receptors (GPCRs) have emerged as novel targets for diabetes therapies, we used this cell line to screen an siRNA library targeting all known mouse GPCRs. We identified several known GPCR regulators of insulin secretion as regulators of the insulin promoter. One of the top positive regulators was Gpr27, an orphan GPCR with no known role in beta cell function. We show that knockdown of Gpr27 reduces endogenous mouse insulin promoter activity and glucose stimulated insulin secretion. Furthermore, we show that Pdx1 is important for Gpr27's effect on the insulin promoter and insulin secretion. Finally, the over-expression of Gpr27 in 293T cells increases inositol phosphate levels, while knockdown of Gpr27 in MIN6 cells reduces inositol phosphate levels, suggesting this orphan GPCR might couple to Gq/11. In summary, we demonstrate a MIN6-based siRNA screening system that allows rapid identification of novel positive and negative regulators of the insulin promoter. Using this system, we identify Gpr27 as a positive regulator of insulin production.


Subject(s)
Insulin-Secreting Cells/metabolism , Insulin/genetics , Promoter Regions, Genetic , RNA, Small Interfering/genetics , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Animals , Cell Line , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/metabolism , Gene Expression Regulation , Gene Knockdown Techniques , Glucose/metabolism , HEK293 Cells , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Inositol Phosphates/metabolism , Insulin/metabolism , Insulin Secretion , Insulin-Secreting Cells/cytology , Mice , RNA, Small Interfering/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism
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