United States

Osteoporosis is a prevalent disease with substantial morbidity/mortality among the aging population. Due to gaps in knowledge, current therapeutics are limited in their ability to prevent degeneration of bone while also stimulating formation of new bone. Teriparatide (PTH (1-34)) and its analogs PTHrP (1-36) and abaloparatide (ABL), have been utilized for treatment of osteoporosis but have significant limitations in efficacy over long-term use. Research from our laboratory has shown PTH (1-34), PTHrP (1-36), and ABL exert time and dose-dependent differential responses in the osteoblast, leading us to hypothesize they may also differentially modulate the osteoblast transcriptome. In this study we show that treatment of mouse calvarial osteoblasts with 1 nM of these peptides for 4 h results in differing effects on the osteoblast transcriptome by performing gene enrichment analysis of RNA-Seq data. Genes were selected with a Log2 fold change &gt;1 and a false discovery rate &lt;0.05. These data were analyzed and compiled into heat maps for each peptide and smear/volcano plots. RNA-Sequencing revealed that PTH (1-34) regulated 367 genes, 194 were unique; PTHrP (1-36) regulated 117 genes, 15 were unique; ABL regulated 179 genes, 20 were unique. There were 74 genes shared only among PTH(1-34) and ABL; 16 genes shared only among PTH (1-34) and PTHrP; and 83 genes shared among all three peptides. Data collected show pathway-specific differences including, 1) cAMP/PKA, 2) Wnt/β-catenin, 3) Transcriptional regulation 4) Inflammatory response 5) Transmembrane transport, 6) Metabolism, and 7) NF-kB. Further analysis of the data illuminated that the three peptides increased Vitamin D receptor (VDR) and Cbp/p300-interacting transactivator 1 (CITED1) mRNAs similar to RankL expression. These findings were confirmed via qRT-PCR of additional cultured samples of mouse calvarial osteoblasts, treated with 1 nM of PTH (1-34), PTHrP (1-36), and ABL for 4 h prior to harvest. RankL mRNA, VDR mRNA, and CITED1 mRNA were measured for each sample and statistical differences were analyzed via Kruskal Wallis p&lt;0.05. Additionally, we analyzed mRNA levels of several genes of interest, including WNT7b, WNT11, TCF7, SFRP4, FZD5, PP2R2A, and DVL3 mRNA. Pathway analysis and subsequent qRT-PCR confirmation has shown that PTH (1-34) and ABL lead to a significant increase in Wnt11 mRNA, while PTHrP (1-36) does not. Our findings highlight the complexity of the genetic and functional events triggered by PTH (1-34) and its analogs. Many studies have examined PTH signaling in the osteoblast/osteocyte; ours is the first to examine global effects of these peptides on the osteoblast transcriptome. Further delineation of which signaling events are attributable to PTH (1-34), PTHrP (1-36), and ABL exclusively and which are shared among all three will further our understanding of the effects these peptides have on the osteoblast and lead to refinement of PTH-derived treatments for osteoporosis.

ENDO 2022

Comparison of the Effects of PTH (1-34), PTHrP (1-36) and Abaloparatide (ABL) on the Murine Osteoblast Transcriptome

**Program**
ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

This year’s education program features:
more than 100 in-person and virtual sessions with another 70+ in-person only sessions;
six plenary sessions including plenaries on Envisioning Future Therapies for Type 1 Diabetes and Addressing Challenges in Fertility and Reproduction;
over 20 Meet the Professor sessions presented both in-person and live-streamed virtually and another 20+ presented live in Atlanta;
four Basic Science Pathways, including Diabetes and Metabolism; Neuroendocrinology; Nuclear Receptors and Signaling; and Reproductive Endocrinology; and
more than 30 oral sessions and a robust virtual poster hall for accepted scientific abstracts.

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As you build your schedule during the conference, you can select the sessions that are tagged with 'Livestreamed', so you can view virtually. These sessions are being livestreamed, so they are available in real time to our virtual audience. In addition, our in-person and livestreamed sessions will be recorded, so you can access them on demand after 72 hours. We will have over 200 hours available on demand for Standard and Premium pass registrants.

Product theaters and the Communications and Career Center will also include hybrid content delivery.

**Plenaries**
Spanning the spectrum of science, clinical care, and social implications, an outstanding slate of world-renowned speakers will showcase the most cutting-edge advances on research and medicine.

**Symposia**
Our ENDO 2022 lineup of sessions offers a unique opportunity to dive into topics from nearly every major therapeutic area of endocrinology and hormone science. Listen, learn, and be inspired by world-class faculty who are at the forefront of scientific discovery and high-quality health care.

**Basic Science Pathways**

These special “meeting-within-a-meeting” tracks integrate focused discussions, poster sessions, and networking opportunities to provide you with the latest research in:
Diabetes and Metabolism
Neuroendocrinology
Nuclear Receptors and Signaling
Reproductive Endocrinology

**Meet the Professor & Meet the Scientist**
Discover insights from experts on treating a variety of endocrine conditions and emerging science in the field of endocrinology, and learn the latest in patient care and scientific research.

ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

Osteoporosis is a prevalent disease with substantial morbidity/mortality among the aging population. Due to gaps in knowledge, current therapeutics are limited in their ability to prevent degeneration of bone while also stimulating formation of new bone. Teriparatide (PTH (1-34)) and its analogs PTHrP (1-36) and abaloparatide (ABL), have been utilized for treatment of osteoporosis but have significant limitations in efficacy over long-term use. Research from our laboratory has shown PTH (1-34), PTHrP (1-36), and ABL exert time and dose-dependent differential responses in the osteoblast, leading us to hypothesize they may also differentially modulate the osteoblast transcriptome. In this study we show that treatment of mouse calvarial osteoblasts with 1 nM of these peptides for 4 h results in differing effects on the osteoblast transcriptome by performing gene enrichment analysis of RNA-Seq data. Genes were selected with a Log2 fold change >1 and a false discovery rate <0.05. These data were analyzed and compiled into heat maps for each peptide and smear/volcano plots. RNA-Sequencing revealed that PTH (1-34) regulated 367 genes, 194 were unique; PTHrP (1-36) regulated 117 genes, 15 were unique; ABL regulated 179 genes, 20 were unique. There were 74 genes shared only among PTH(1-34) and ABL; 16 genes shared only among PTH (1-34) and PTHrP; and 83 genes shared among all three peptides. Data collected show pathway-specific differences including, 1) cAMP/PKA, 2) Wnt/β-catenin, 3) Transcriptional regulation 4) Inflammatory response 5) Transmembrane transport, 6) Metabolism, and 7) NF-kB. Further analysis of the data illuminated that the three peptides increased Vitamin D receptor (VDR) and Cbp/p300-interacting transactivator 1 (CITED1) mRNAs similar to RankL expression. These findings were confirmed via qRT-PCR of additional cultured samples of mouse calvarial osteoblasts, treated with 1 nM of PTH (1-34), PTHrP (1-36), and ABL for 4 h prior to harvest. RankL mRNA, VDR mRNA, and CITED1 mRNA were measured for each sample and statistical differences were analyzed via Kruskal Wallis p<0.05. Additionally, we analyzed mRNA levels of several genes of interest, including WNT7b, WNT11, TCF7, SFRP4, FZD5, PP2R2A, and DVL3 mRNA. Pathway analysis and subsequent qRT-PCR confirmation has shown that PTH (1-34) and ABL lead to a significant increase in Wnt11 mRNA, while PTHrP (1-36) does not. Our findings highlight the complexity of the genetic and functional events triggered by PTH (1-34) and its analogs. Many studies have examined PTH signaling in the osteoblast/osteocyte; ours is the first to examine global effects of these peptides on the osteoblast transcriptome. Further delineation of which signaling events are attributable to PTH (1-34), PTHrP (1-36), and ABL exclusively and which are shared among all three will further our understanding of the effects these peptides have on the osteoblast and lead to refinement of PTH-derived treatments for osteoporosis.

technical paper

Background: A 24-hour urine collection is routinely obtained in the workup of primary hyperparathyroidism (PHPT) to measure calcium and creatinine excretion, rule out familial hypocalciuric hypercalcemia, and guide diagnosis and management. Although hypercalciuria is expected and frequently observed in PHPT, hypocalciuria and/or a low urine calcium (uCa) to creatinine clearance ratio (UCCCR) may occur. Variables including race, age, renal function, and 25-OH vitamin D levels can affect urinary calcium excretion. Given the utility of obtaining urine calcium excretion in patients with PHPT, we performed a retrospective analysis on patients with PHPT to evaluate the effect of clinical variables on urinary calcium excretion, and to test the hypothesis that hypocalciuria may delay parathyroidectomy in PHPT.Methods: We retrospectively reviewed charts of patients with PHPT with available 24-hour uCa who underwent successful parathyroidectomy at our institution between 2009 and 2021. We extracted available demographic, clinical, and laboratory data including first available uCa and creatinine excretion prior to parathyroidectomy, age, gender, ethnicity, BMI, eGFR, serum calcium, phosphorus, PTH, 25-hydroxyvitamin D, and 24-hour sodium excretion. The association between these parameters and uCa and UCCR was assessed in univariate and multivariate models. We compared characteristics of PHPT patients with vs. without hypocalciuria, and duration between uCa measurement and parathyroidectomy.Results: 643 PHPT patients were included in this analysis. 8.4% of patients had a 24-hr uCa excretion rate below 100mg/day, and 18.7% had a UCCR<1%.Compared to PHPT patients with UCCCR≥1%, those with UCCCR<1% had a significantly higher proportion of African Americans (22.2% vs 9.1%; p=0.0029), a lower mean serum calcium (p=0.0027), and 25-hydroxyvitamin D (p=0.035). In multivariate analysis, gender, race, serum calcium, serum 25-OH-vitamin D, and 24-hour urine sodium were all significant predictors of UCCCR. The UCCCR<1% group had a significantly longer median time from uCa measurement to parathyroidectomy [8.4 (Interquartile range IQR: 3.3-29.4) vs. 4.2 (IQR: 2.4-8.7) months, p<0.001).Compared to PHPT patients with 24-hr uCa≥100 mg/day, those with 24-hr uCa<100 mg/day had significantly lower mean serum calcium (p=0.002), eGFR (p<0.0001), urine sodium excretion (p=0.036), and significantly greater serum phosphorus (p=0.0085) and PTH (0.0078). In multivariate analysis, race, BMI, serum calcium, eGFR and urine sodium were all significant predictors of 24-hr uCa. The uCa<100 mg/day group had a significantly longer median time from uCa measurement to parathyroidectomy [9.7 (IQR: 3.3-32.2) vs. 4.2 (IQR: 2.4-8.8) months, p<0.001).Conclusions: Although elevated uCa is used as a diagnostic marker and indication for parathyroidectomy in PHPT, hypocalciuria and UCCCR<1% are relatively common in PHPT patients, and associated with a significant delay in parathyroidectomy. Intrinsic (race, gender) and extrinsic (vitamin D status, renal function, sodium intake/excretion) factors are all determinants of calcium excretion in PHPT.

Hypocalciuria is Common and Associated with Delayed Parathyroidectomy in Patients with Primary Hyperparathyroidism

PTH peptides can be effective in normalizing blood calcium (Ca++) levels in cases of hypoparathyroidism (HP), but long-term effects on bone are not well understood. AZP-3601 is a long-acting PTH/PTHrP(1-36) analog currently being investigated as a potential treatment option for HP. As compared to conventional PTH1-34, AZP-3601 binds with higher affinity to the R0 conformation of the PTH receptor and thereby induces more sustained signaling responses in cells as well as more prolonged calcemic responses in vivo, despite having a very short circulating half-life. Using the TPTX rat model of HP, we investigated whether the distinct mode-of-action used by AZP-3601 results in different down-stream effects in bone than those induced by PTH1-34, when the peptides are administered long-term (31 days) either by daily sc injection (AZP-3601 and PTH1-34) or by continuous infusion (PTH1-34), and at doses aimed to optimally normalize blood Ca++ levels.  Methods: Male TPTX rats (strain S-D, age 9 weeks, and 2 weeks post TPTX surgery) were injected daily with either AZP-3601 at a dose of 0.7 nmol/kg or PTH1-34 at a dose of 50 nmol/kg, or were continuously infused via Alzet mini-pump with PTH1-34 at a dose of 3.0 nmol/kg/day. Vehicle treatments and sham surgery rats were used as controls. Tail-vein blood was collected on days 1, 7 14, 21 and 28 at 6 hours post-injection and analyzed for total Ca++. Rats were euthanized on day 31 (24 hours after last injection) and blood and femurs collected for analysis.  Results: All peptide-treatments increased serum Ca++ levels on days 7-21 to within or near the normal range (8.8-11.7 mg/dL versus 10.8-11.9 mg/dL in sham-operated rats) and to levels significantly higher than those in vehicle-treated TPTX rats (6.9-7.9 mg/dL, P<0.01). Bone mCT analysis revealed that relative to TPTX-vehicle controls, PTH1-34 continuous infusion significantly decreased (p<0.001) distal femur trabecular bone volume relative to tissue volume (BV/TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th), while PTH1-34 daily injection significantly increased (P<0.001) distal femur BV/TV, Tb.Th and Tb.N, as well as mid-femur cortical thickness (Ct.Th, P<0.001). In contrast, AZP-3601 daily injection caused no change in trabecular or cortical bone parameters. Serum levels of the bone formation marker P1NP were significantly increased by daily injection of PTH1-34 (P<0.001 vs. TPTX-vehicle) but were not changed by other treatments.  Conclusion: With long-term (31 days) treatment and at doses that similarly normalized serum Ca++ in TPTX rats, effects on bone were catabolic for PTH1-34 by continuous infusion, anabolic for PTH1-34 by daily injection, and neutral for AZP-3601 by daily injection. The distinct MOA used by AZP-3601 may thus result in less of an impact on bone than either daily injection or sustained, continuous delivery of PTH(1-34), when used as a long-term treatment for HP.

AZP-3601, a Long-Acting Parathyroid Hormone (PTH) Analog, Normalizes Blood Calcium in Thyroparathyroidectomized (TPTX) Rats Without Causing Deleterious Changes in Bone

Late Breaking Tools and Targets in Basic Endocrinology Research

Obesity is a major risk factor for developing metabolic diseases. Adipose tissue stores excess calories during obesity. Nuclear receptor Farnesoid X receptor (FXR) is expressed in various tissues and transcriptionally regulates lipid and glucose metabolism. However, the role of FXR in regulating adipose expansion is unknown. We showed that Fxr transcript is expressed in mature adipocytes isolated from both white (WAT) and brown (BAT) adipose tissues. In adipocyte-specific Fxr knockout (Ad-FxrKO) mice, we found enlarged adipocytes and decreased expression of lipolytic genes upon 60% high fat diet (HFD) or high fat/high sucrose western diet challenge. Transcriptomic analysis of WAT from Ad-FxrKO and control mice upon chow or HFD revealed that many genes involved in glucose and lipid homeostasis are misregulated in Ad-FxrKO mice with distinct patterns under normal and obese conditions. Since WAT stores fat and BAT generates heat, we examined both these depots in Ad-FxrKO mice and found reductions in the expression of mitochondrial genes and energy expenditure. Intriguingly, we found ectopic fat accumulation in the liver and systemic glucose intolerance in Ad-FxrKO compared to the control mice during obesity. Overall, these findings implicate that FXR may regulate lipid metabolism and mitochondrial functions in the adipose tissue, which consequently impacts whole-body energy homeostasis.

FXR Regulates the Expansion and Metabolic Function of Adipose Tissue upon Caloric Excess

Maternal undernutrition (MU) during fetal development profoundly effects offspring metabolism, resulting in either blunting or premature shifting of the neonatal leptin surge, and can cause metabolic dysfunction in adulthood. We developed a mild MU model in FVB mice, where dams received 20% less caloric intake (underfed) compared to control, ad libitum fed (Fed) mice. As previously reported, underfed progeny exhibited a leptin surge that was 3 days earlier (PND8) than controls (PND11) and PND1 GH levels were 354% higher. However serum IGF-1 was 55% lower (p=0.027) at PND16 in underfed progeny, resulting in delayed puberty and growth deficiencies which persisted in the male offspring. At 3 months, fed and underfed offspring received either a 45% HFD or 10% fat (Control) diet for 16 weeks, resulting in a weight gain (>12 g) in progeny from fed dams. However, underfed females gained only 8.74 g on the HFD, [lower than fed females (p=0.03)]. More surprisingly, underfed males showed no weight gain in response to the HFD. The study was replicated, yielding the same results.
We hypothesize that resistance to HFD induced weight gain by underfed males may be due to altered transcriptome expression. We investigated this unusual response through RNA sequencing of the pituitary, fat and liver of both fed and underfed male progeny. Pituitary and liver transcriptome analysis are ongoing. This presentation focuses on changes in the fat transcriptome.
Fat from Fed HFD males had 592 upregulated and 153 downregulated transcripts when compared to controls. Strikingly, fat of underfed HFD males had only 3 upregulated and 3 downregulated transcripts when compared to underfed controls. To ascertain potential protective influences, transcripts in the fat of Fed HFD males were compared with those of underfed HFD males. Prelid3a, Peg3, and Thbs1 were differentially expressed in underfed HFD males compared to Fed HFD males. Prelid3a, (involved in mitochondrial lipid metabolism and transport) was downregulated 2.9 fold (0.035 adj. P) in underfed HFD fat. Peg3,(involved in fat accumulation when decreased) was increased in underfed HFD fat by 2.4 fold (0.0297 adj. P). Thbs1 was also increased by 2.7 fold (0.0476 adj. P) in underfed HFD fat. Thbs1 contributes to HFD weight gain when increased, but can also improve hepatic steatosis and promote a protective ER stress response. Pde3b, found to be protective against obesity, was reduced 1.4 fold (0.002 adj. P) in Fed HFD males, but unchanged in underfed HFD males.
These data indicate that mild undernutrition causes a premature leptin surge, marked by sex-specific differences in offspring growth and transcriptome regulation in the fat of male offspring that is beneficial to blunting HFD induced weight gain.

Mild Maternal Undernutrition Results in Transcript Regulation in the Fat of Male Offspring that Correlates with Resistance to High Fat Diet Induced Weight Gain

Neurons that co-express the orexigenic agouti-related protein (AgRP) and neuropeptide Y (NPY) are indispensable for normal feeding behavior. Firing activities of AgRP/NPY neurons dynamically fluctuate with energy status and coordinate appropriate feeding behavior to meet nutritional demands. We previously demonstrated that asprosin, a recently discovered fasting-induced glucogenic and orexigenic hormone, crosses the blood-brain barrier and directly activates the orexigenic AgRP/NPY neurons via a cyclic cAMP-dependent pathway. However, intrinsic mechanisms on how asprosin regulates AgRP/NPY neural activities during the fed-to-fasted transition are not fully understood. In the satiated mice with low circulating levels of asprosin, we found that AgRP/NPY neurons expressed high levels of the small conductance calcium-activated potassium channel 3 (SK3) and were inhibited by SK3-mediated potassium currents. Conversely, in 24 hour-fasting mice with high levels of asprosin, AgRP/NPY neurons expressed low levels of SK3 and were activated via inhibition of SK3-mediated potassium currents. Notably, the stimulatory effects of asprosin on AgRP/NPY neurons were blunted in mice with SK3 selectively deleted in the AgRP/NPY neurons, further supporting a mediating role of SK3.
Moreover, we also identified protein tyrosine phosphatase receptor δ (Ptprd), a membrane-bound phosphatase receptor, as the orexigenic asprosin-receptor. Deletion of Ptprd in AgRP/NPY neurons abolished asprosin's stimulatory effect on SK3-mediated currents and protected mice from diet-induced obesity.
Lastly, increased circulating levels of asprosin promoted hyperphagia, body weight gain, and glucose intolerance. On the other hand, anti-asprosin antibody treatment decreased food intake and body weight by inhibiting AgRP/NPY and other orexigenic hypothalamic neurons via SK currents stimulation.
In summary, our data support a model that asprosin acts on Ptprd expressed by AgRP/NPY neurons to inhibit SK3 current, further activating AgRP/NPY neurons to increase food intake and body weight. Our findings also provided preclinical evidence that asprosin-neutralizing antibodies could be used to treat obesity.

Hunger hormone asprosin activates orexigenic neurons via SK currents

Free fatty acid receptor 2 and free fatty acid receptor 3 (FFA2/3) are two highly similar G protein-coupled receptors belonging to the free fatty acid receptor family. Their ligands are short-chain fatty acids (SCFAs), which are key nutrients that play a diverse role in physiological function, including the regulation of metabolic homeostasis and glycemic control. FFA2/3 are broadly expressed in a multitude of tissues including the intestine, pancreas, adipose and central nervous system, where they contribute to metabolic homeostasis via a summation of tissue-specific effects. Consequently, FFA2/3 have been identified as a potential drug target for metabolic diseases including obesity and type-2 diabetes.
Both FFA2 and FFA3 are highly expressed within the intestinal epithelium – the major site of SCFA generation – and have been identified in hormone-secreting enteroendocrine cells as well as intestinal epithelial cells. However, due conflicting data, the respective roles of FFA2/3 within the intestine and their effects on physiology and metabolism are still largely unclear. Previous in vivo studies involving this receptor have largely relied on global knockout mouse models, making it difficult to isolate their effects in the intestine. To overcome this challenge, we generated a novel intestine-specific knockout mouse model for FFA2 and FFA3 individually, utilizing Cre-mediated recombination under the expression of the villin promoter. Here, we report the first in vivo characterization of FFA2/3 in the intestine and reveal novel insights into receptor function.
Following model validation, we conducted a general metabolic assessment of male Villin-Cre-FFA2 (Vil-FFA2) and Villin-Cre-FFA3 (Vil-FFA3) mice on standard chow and observed no major congenital or time-dependent defects. Because dietary changes are known to alter gut microbial composition, and thereby SCFA production, a pilot study was performed on male Vil-FFA2 and Vil-FFA3 mice and their littermate controls to probe for a phenotype on a high-fat, high-sugar "western diet." Mice were placed on either a low-fat control diet (CD) or western diet (WD) at 10 weeks of age and metabolically profiled for 25 weeks. We found that both Vil-FFA2 and Vil-FFA3 mouse strains were largely protected from diet-induced obesity and had significantly lower fat mass as well as adipose hypertrophy. Additionally, both mouse strains had reduced intestinal inflammation and improved glucose homeostasis. These differences were driven by lower food intake in the Vil-FFA2 strain only. Our findings suggest a novel role of FFA2/3 in mediating the metabolic consequences of a western diet – a state of high inflammation, dysbiosis and metabolic stress. Moreover, these data support an intestine-specific role of FFA2/3 in whole-body metabolic homeostasis and in the development of adiposity and hyperglycemia.

Intestinal FFA2 and FFA3 Mediate Obesogenic Effects in Mice on a Western Diet

Adipogenesis is a complex and tightly regulated process that plays a vital role in multiple aspects of human health.  The molecular mechanisms regulating adipogenesis are incompletely understood, although key facets of the signaling and regulatory pathways have been defined.  ADP-ribosylation – the process whereby ADP-ribose moieties are covalently transferred from NAD+ to substrate proteins – has been shown to control multiple components of the adipogenic regulatory machinery, including the transcription factor C/EBPb. In order to explore the role of mono(ADP-ribosyl)transferases (MARTs) during adipogenesis, we used an siRNA-mediated knockdown screen in mouse 3T3-L1 preadipocytes to determine which MARTs are required for the differentiation of the preadipocytes into mature adipocytes. This screen identified PARP7 the primary candidate. Using a variety of cell-based and biochemical assays, we have identified key aspects of PARP7's involvement throughout adipogenesis. We observed dynamic localization of PARP7 during adipogenesis, moving from the nucleus in early adipogenesis to the cytosol during mid to late adipogenesis. The requirement for PARP7, as determined by knockdown, was most evident in early stages of adipogenesis.  Knockdown of PARP7 was rescued by a PPAR g agonist, suggesting that PARP7 acts before the onset of the later stages of adipogenesis leading to mature adipocytes. Together, these findings led us to hypothesize that PARP7 plays a critical role in the nucleus during early adipogenesis. Preliminary data have revealed that PARP7 binds to C/EBPb, a key "first wave" transcription factor that drives adipogenesis, during a time when C/EBPb is poly(ADP-ribosyl)ated by PARP1 in the nucleus. Ongoing investigations are exploring: (1) the mechanisms by which PARP7 binds to C/EBPb; (2) the impact of PARP7 binding on C/EBPb stability; and (3) the biological outcomes of PARP7-C/EBPb interactions. Collectively, these studies provide an avenue to better understand the role of PARP7 in the regulation of adipogenesis.
This work is supported by a grant from the NIH/NIDDK (R01 DK069710) and funds from the Cecil H. and Ida Green Center for Reproductive Biology Sciences Endowment to W.L.K., and a predoctoral fellowship from the American Heart Association to M.S.S.

Exploring the Role of PARP7 in Adipogenesis

Background: Skeletal muscle is one of to the most dynamic and plastic tissues. The capacity to regenerate arises from adult muscle stem cells termed myogenic progenitors (MPs) that are activated in response to injury and differentiate to replenish damaged muscle fibers. The age-related loss of muscle mass is reflected in the reduced regenerative potential of MPs. The underlying mechanisms are still unclear and insights into alterations in adult muscle stem cells during ageing are urgently needed. We have previously identified a novel muscle-specific microRNA, miR-501-3p, that is enriched in activated MPs. Pharmacological inhibition of miR-501 during muscle regeneration promoted small-diameter neofibers. Here, we provide evidence for the regulation of miR-501 in skeletal muscle during aging and its effect on newly formed myofibers using genetic deletion in mice.Methods: We analysed the regulation and upstream signalling pathways of miR-501 in primary myoblasts from mice and humans and in skeletal muscle from young and aged mice. We generated a novel Cre-loxP mouse model, which allowed for global and MP-specific deletion of miR-501. Skeletal muscle regeneration was induced using cardiotoxin (CTX) and assessed using immunofluorescence and transcriptomic analysis.Results: miR-501 was upregulated in muscle cells by the paracrine growth factors platelet derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) through the Janus tyrosine kinase (JAK)-signal transducer and activator of transcription-3 (STAT3) signalling axis. Expression levels of miR-501 as well as its upstream regulators Pdgf and Vegf were significantly reduced in skeletal muscle from aged mice (22 months) compared to young mice (3 months) by 67.8% (p<0.001), 40.0% (p<0.01) and 36.3% (p<0.05), respectively. Genetic deletion of miR-501 in mice resulted in muscle fibers with decreased size in adult skeletal muscle and in newly formed myofibers during muscle regeneration. RNA-seq of regenerating muscle annotated a critical role for miR-501 in the organisation of muscle filaments. We identified a miR-501-dependent sarcomeric gene signature that involves genes such as myosin heavy chains, titin and tubulin polymerization-promoting protein. This was confirmed in vitro in myotubes from mice and humans in which miR-501 was deleted either genetically or pharmacologically using antagomirs. Altered sarcomeric gene expression correlated with an increased sarcomere length in myotubes lacking miR-501 (2.49 ± 0.08μm vs 3.22 ± 0.15μm, p<0.01).Conclusion: Our data indicate that paracrine growth factor signalling contributes to muscle fiber formation via miR-501 in myogenic progenitor cells and its effect on the sarcomere. Restoring the Pdgf+Vegf/miR-501 axis might have the potential to improve muscle formation during aging.

Paracrine Growth Factor Signaling Regulates Muscle Regeneration in Aged Mice via microRNA 501

Background and purpose of the study
The overall goal of this study is to develop precision-targeted therapies for type 1 diabetes (T1D) by blocking the presentation of pancreatic antigens to T cells using an innovative translational approach. There is currently no curative therapy for T1D and the only available treatment is insulin replacement, which – although being life-saving – remains problematic and can be associated with often fatal high or low glucose levels. We hypothesize that the presentation of pathogenic pancreatic peptides to T cells within the HLA-DQ8 pocket is a key trigger in T1D, and that blocking the peptide binding to this pocket can be harnessed to treat/prevent the autoimmune response targeting beta cells in T1D. We propose to block antigen presentation to treat/prevent autoimmunity in T1D using retro-inverso D-amino acid peptides (RIPs). To test our hypothesis, we used the following tools: recombinant human HLA-DQ8, human B cells homozygous for HLA-DQ8, humanized mice expressing human HLA-DQ8, NOD mice, and peripheral blood mononuclear cells (PBMCs) isolated from new onset HLA-DQ8 T1D patients. All of them represent exquisite translational instruments to evaluate in vitro, ex vivo, and in vivo the functional role of our RIPs in preventing beta cell destruction.
Summary of results
We have identified a RIP (RI-CT) that inhibits InsB: 9-23 binding to recombinant HLA-DQ8 molecule, as well as its binding to HLA-DQ8 expressed on a human B-cell line. Specifically, RI-CT averted T-cell activation in a mixed lymphocyte reaction containing human DQ8 cells loaded with InsB: 9-23 peptide and murine T-cells expressing a human TCR specific for the InsB: 9-23–DQ8 complex. These results were also confirmed in humanized transgenic B6-DQ8 mice (KO for murine MHC-II) both ex vivo and in vivo, as shown by decreased production of pro-inflammatory cytokines (including IL-2 and IFN-γ) and reduced lymphocyte proliferation. Moreover, RI-CT prevents autoimmune diabetes in NOD mice as demonstrated by a significant decrease in pancreatic immune cell infiltration in treated mice versus controls. Importantly, at 25 weeks of age 70% of RI-CT treated mice were protected from the development of diabetes, while as expected 80% of control NOD mice had developed the disease. Interestingly, RI-CT injection also altered T-cell subpopulations in NOD mice significantly decreasing CD8 expression and significantly increasing Foxp3 levels in treated mice. Of note, RI-CT significantly inhibits InsB: 9-23-mediated lymphocyte activation in peripheral blood mononuclear cells isolated from new onset DQ8-T1D patients.
Conclusions
In summary, we discovered a RIP that blocks InsB: 9-23 binding to HLA-DQ8 and its presentation to T-cells averting beta cells destruction, and that delays T1D in NOD mice. These data set the stage for using our approach of blocking antigen presentation by RIP as a novel therapeutic approach for autoimmune diseases in general.

Comparison of the Effects of PTH (1-34), PTHrP (1-36) and Abaloparatide (ABL) on the Murine Osteoblast Transcriptome

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Hypocalciuria is Common and Associated with Delayed Parathyroidectomy in Patients with Primary Hyperparathyroidism

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