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Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin-producing β-cells. While islet transplantation is a promising alternative to insulin therapy for T1D treatment, finding ideal transplant sites that are readily accessible, exhibit glucose regulation, are immune-privileged, and promote vascularization for islet survival has remained a challenge. A novel site which exhibits these traits is the brown adipose tissue (BAT). BAT functions as a thermogenic adipose tissue that maintains body temperature and regulates metabolism. We hypothesize that murine islets transplanted into BAT will display normal islet graft and BAT function while providing immunological protection. Syngeneic transplant of 250 NOD.scid islets into the BAT of streptozotocin-treated diabetic NOD.scid mice restored euglycemia for over ten weeks, which was comparable to kidney capsule transplant controls. BAT histology and mRNA analysis assessed ten weeks after transplantation showed engrafted islets maintain expression of hallmark hormones, like insulin and glucagon, and critical transcription factors. Evaluation of BAT function after transplantation revealed no impairments in glucose and insulin tolerance, cold tolerance, or whole-body energy expenditure. Flow cytometry analysis displayed an enrichment of immunomodulatory T regulatory cells and anti-inflammatory M2 macrophages in BAT compared to kidney, possibly allowing greater immunoprotection for engrafted islets. Strikingly, adoptive transfer and allogeneic transplant studies revealed that islets transplanted into BAT showed significant delays in autoimmune and allograft rejection compared to islets transplanted under the kidney capsule. Overall, our data suggest BAT as a novel and efficacious site for islet transplantation to potentially combat T1D. Disclosure J. Kepple: None. J. Barra: None. C. S. Hunter: None. H. M. Tse: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (DK099550 to H.M.T.), (DK111483 to C.S.H.), (DK126456 to H.M.T., C.S.H.), (F31DK121414 to J.K.); JDRF (SRA-2016-270-S-B, 2-SRA-2019-692-S-B to H.M.T.); National Institute of General Medical

Brown adipose tissue (BAT) is critical for mammalian thermogenesis. BAT utilizes fatty acids and glucose for heat production via mitochondrial uncoupling and is thus an attractive therapeutic target for combatting obesity. Exploiting the energy uncoupling capacity of this tissue will require a greater understanding of underlying BAT transcriptional mechanisms. Our prior work using a global deficiency model supported a novel role for the LIM domain-binding protein 1 (Ldb1) transcriptional co-regulator in regulating BAT function. However, direct roles for Ldb1 expressed in brown adipocytes has not been tested. Ldb1 acts as a scaffold to promote the assembly of transcriptional complexes and is important for the development and function of several metabolic tissues. We hypothesized that Ldb1 maintains BAT thermogenic function through direct regulation of thermogenic genes, like Ucp1. Loss of Ldb1 in primary brown adipocytes resulted in reduced expression of BAT-selective genes including Ucp1 and Elovl3 and the inability to induce Ucp1 expression upon Beta-3 adrenergic receptor agonism. This result was replicated using a siLdb1-transfected Ucp1-expressing cell line. To test Ldb1 roles on systemic thermogenesis, we developed a mouse model, termed Ldb1ΔBAT, where Ldb1 was reduced in thermogenic adipocytes using a Ucp1-driven Cre recombinase. Similar to our in vitro observations, Ldb1ΔBAT mice had reduced mRNAs of key BAT genes, including Ucp1 and Cidea. Through indirect calorimetry and a cold challenge, we uncovered significant impairments in energy expenditure and cold intolerance in Ldb1ΔBAT mice. To assess the transcriptional mechanisms underlying Ldb1 impacts on BAT thermogenesis, we conducted chromatin immunoprecipitation and found Ldb1 occupied upstream regulatory domains of Ucp1 in chromatin prepared from a BAT cell line and primary tissue. Overall, this work supports that Ldb1 is required for the thermogenic function of brown adipocytes. Disclosure J. Kepple: None. Y. Liu: None. T. Kim: None. K.M. Habegger: Consultant; Self; Glyscend, Inc. Research Support; Self; Daiichi Sankyo, Novo Nordisk Inc. M. Young: None. C.S. Hunter: None. Funding National Institutes of Health (R01DK111483, F31DK121414, T32GM109780)

Type 1 diabetes (T1D) results in hyperglycemia, increased risk for secondary complications, and life-long dependence on exogenous insulin. Islet transplantation is a promising alternative to insulin therapy for T1D treatment. However, long-term function of islets transplanted into the FDA approved hepatic portal vein remains low, with much of the islet mass being lost early after transplantation due to immune responses and drug toxicity. These challenges have prompted a search for alternative transplantation sites that are readily accessible, immune-privileged, offer graft retrievability, and promote islet survival. A novel site that exhibits these traits is brown adipose tissue (BAT), which is highly vascularized, innervated, and contains immunomodulatory immune cells. BAT functions to maintain thermogenesis and can regulate metabolic homeostasis. We hypothesize that islet engraftment in BAT can restore euglycemia in diabetic mice, promote revascularization, and delay immune rejection. Syngeneic transplantation of 250 NOD.scid islets into the BAT of streptozotocin (STZ)-treated diabetic NOD.scid mice restored euglycemia for over two months, similar to islets transplanted under the pre-clinical kidney capsule transplant site. Islet function was also comparable across transplant groups as measured by a glucose tolerance test. BAT thermogenic function during a cold challenge showed no difference in cold tolerance after transplantation. Histology and mRNA analysis revealed BAT engrafted islets expressed islet hormones, functional islet transcription factors, and CD31+ vascularization at the islet graft site. Overall, our data suggest BAT is a novel and efficacious site for islet transplantation. Future islet transplant studies will determine if the inherent properties of BAT can extend the longevity of transplanted islets by investigating the immunomodulatory capacity of BAT-resident mesenchymal stem cells and immune cells, compared to current transplantation sites. Disclosure J. Barra: None. J. Kepple: None. H.M. Tse: None. C.S. Hunter: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK099550); JDRF (SRA-2016-270-S-B, 2-SRA-2019-692-S-B); National Institute of General Medical Sciences (T32GM008111)

Brown adipose tissue (BAT) is critical for thermogenesis and glucose/lipid homeostasis. BAT utilizes fatty acids and glucose for heat production via mitochondrial uncoupling, and is thus an attractive therapeutic target for combatting obesity. Expanding the energy uncoupling capacity of this tissue requires a greater understanding of underlying BAT transcriptional mechanisms. We recently reported on a transcriptional co-regulator, LIM domain binding protein 1 (Ldb1), which appears to have novel roles in BAT biology. Ldb1 acts as a dimerized scaffold allowing for the assembly of transcriptional complexes and is important for the development and function of many metabolic tissues, including the brain and pancreatic islets. However, roles for BAT-expressed Ldb1 have not been elucidated. We set out to test the hypothesis that Ldb1 directly impacts BAT development and function. Our in vitro studies have shown that primary brown adipocytes lacking Ldb1 have reductions in BAT-selective genes including Ucp1 and Elovl3 and are unable to induce Ucp1 expression upon agonist treatment, a result similarly observed in the X9 Ucp1-expressing cell line. To test Ldb1 roles in vivo, we developed a mouse model, termed Ldb1ΔBAT, in which Ldb1 was reduced in thermogenic adipocytes using a Ucp1-driven Cre recombinase. The Ldb1ΔBAT mice were cold intolerant during a cold challenge, which is supportive of thermogenic defects in the Ldb1-deficient tissue. The knockout mice were also glucose intolerant via intraperitoneal glucose challenge and preliminary data suggest insulin insensitivity, via intraperitoneal insulin tolerance test. Histological analyses of Ldb1ΔBAT BAT depots indicated an alteration in brown adipocyte morphology and potentially intercellular lipid content, suggesting cellular dysfunction. Overall, this work indicates that Ldb1 has a direct role in maintaining thermogenic and metabolic function in BAT. Disclosure J. Kepple: None. Y. Liu: None. C.S. Hunter: None.

Brown adipose tissue (BAT) is critical for thermogenesis and glucose homeostasis. BAT utilizes fatty acids and glucose for heat production via mitochondrial uncoupling, and is thus an attractive therapeutic target for combatting obesity. Exploiting the energy uncoupling capacity of this tissue requires a greater understanding of underlying BAT transcriptional mechanisms. We recently reported on a transcriptional co-regulator, LIM domain binding protein 1 (Ldb1), which appears to have novel roles in BAT biology. Ldb1 acts as a dimerized scaffold allowing for the assembly of transcriptional complexes and is important for the development and function of many tissues, including the brain and pancreatic islets. However, direct roles for BAT-expressed Ldb1 have not been elucidated. We set out to test the hypothesis that Ldb1 directly impacts BAT development and function. We developed a mouse model in which Ldb1 was deleted in thermogenic adipocytes using an Uncoupling protein 1 (Ucp1)-driven Cre recombinase, termed Ldb1δBAT. These knockout mice had reductions in BAT-selective mRNAs, including Ucp1 and Elovl3, a result similarly observed in X9 beige cell lines lacking Ldb1. Ldb1δBAT mice were unable to defend body temperature during a cold challenge, suggesting thermogenic defects. We also observed glucose intolerance in Ldb1δBAT mice via intraperitoneal glucose challenge. Ldb1 deletion in preadipocytes resulted in reduced Ucp1 expression upon induction to mature adipocytes. These data suggest a direct role for Ldb1 in maintaining thermogenic and metabolic function in BAT, as well as a developmental role in preadipocytes. Additional metabolic and transcriptional profiling of the Ldb1δBAT model will interrogate the mechanisms underlying Ldb1 control of BAT function, potentially leading to novel obesity and diabetes therapeutic targets. Disclosure J. Kepple: None. Y. Liu: None. T. Kim: None. G.C. Rowe: None. K.M. Habegger: Consultant; Self; Glyscend, Inc.. Research Support; Self; Glyscend, Inc.. Consultant; Self; Intarcia Therapeutics, Inc.. C.S. Hunter: None.

Islet-1 (Isl-1) is essential for the survival and ensuing differentiation of pancreatic endocrine progenitors. Isl-1 remains expressed in all adult pancreatic endocrine lineages; however, its specific function in the postnatal pancreas is unclear. Here we determine whether Isl-1 plays a distinct role in the postnatal β-cell by performing physiological and morphometric analyses of a tamoxifen-inducible, β-cell–specific Isl-1 loss-of-function mouse: Isl-1L/L; Pdx1-CreERTm. Ablating Isl-1 in postnatal β-cells reduced glucose tolerance without significantly reducing β-cell mass or increasing β-cell apoptosis. Rather, islets from Isl-1L/L; Pdx1-CreERTm mice showed impaired insulin secretion. To identify direct targets of Isl-1, we integrated high-throughput gene expression and Isl-1 chromatin occupancy using islets from Isl-1L/L; Pdx1-CreERTm mice and βTC3 insulinoma cells, respectively. Ablating Isl-1 significantly affected the β-cell transcriptome, including known targets Insulin and MafA as well as novel targets Pdx1 and Slc2a2. Using chromatin immunoprecipitation sequencing and luciferase reporter assays, we found that Isl-1 directly occupies functional regulatory elements of Pdx1 and Slc2a2. Thus Isl-1 is essential for postnatal β-cell function, directly regulates Pdx1 and Slc2a2, and has a mature β-cell cistrome distinct from that of pancreatic endocrine progenitors.

Ldb1 and Ldb2 are coregulators that mediate Lin11-Isl1-Mec3 (LIM)–homeodomain (HD) and LIM-only transcription factor–driven gene regulation. Although both Ldb1 and Ldb2 mRNA were produced in the developing and adult pancreas, immunohistochemical analysis illustrated a broad Ldb1 protein expression pattern during early pancreatogenesis, which subsequently became enriched in islet and ductal cells perinatally. The islet-enriched pattern of Ldb1 was similar to pan-endocrine cell–expressed Islet-1 (Isl1), which was demonstrated in this study to be the primary LIM-HD transcription factor in developing and adult islet cells. Endocrine cell–specific removal of Ldb1 during mouse development resulted in a severe reduction of hormone+ cell numbers (i.e., α, β, and δ) and overt postnatal hyperglycemia, reminiscent of the phenotype described for the Isl1 conditional mutant. In contrast, neither endocrine cell development nor function was affected in the pancreas of Ldb2−/− mice. Gene expression and chromatin immunoprecipitation (ChIP) analyses demonstrated that many important Isl1-activated genes were coregulated by Ldb1, including MafA, Arx, insulin, and Glp1r. However, some genes (i.e., Hb9 and Glut2) only appeared to be impacted by Ldb1 during development. These findings establish Ldb1 as a critical transcriptional coregulator during islet α-, β-, and δ-cell development through Isl1-dependent and potentially Isl1-independent control.