In vivo screen identifies a SIK inhibitor that induces ß cell proliferation through a transient UPR.

It is known that ß cell proliferation expands the ß cell mass during development and under certain hyperglycemic conditions in the adult, a process that may be used for ß cell regeneration in diabetes. Here, through a new high-throughput screen using a luminescence ubiquitination-based cell cycle indicator (LUCCI) in zebrafish, we identify HG-9-91-01 as a driver of proliferation and confirm this effect in mouse and human ß cells. HG-9-91-01 is an inhibitor of salt-inducible kinases (SIKs), and overexpression of Sik1 specifically in ß cells blocks the effect of HG-9-91-01 on ß cell proliferation. Single-cell transcriptomic analyses of mouse ß cells demonstrate that HG-9-91-01 induces a wave of activating transcription factor (ATF)6-dependent unfolded protein response (UPR) before cell cycle entry. Importantly, the UPR wave is not associated with an increase in insulin expression. Additional mechanistic studies indicate that HG-9-91-01 induces multiple signalling effectors downstream of SIK inhibition, including CRTC1, CRTC2, ATF6, IRE1 and mTOR, which integrate to collectively drive ß cell proliferation.

Development of Kinase-Selective, Harmine-Based DYRK1A Inhibitors that Induce Pancreatic Human ß-Cell Proliferation.

DYRK1A has been implicated as an important drug target in various therapeutic areas, including neurological disorders and oncology. DYRK1A has more recently been shown to be involved in pathways regulating human ß-cell proliferation, thus making it a potential therapeutic target for both Type 1 and Type 2 diabetes. Our group, using a high-throughput phenotypic screen, identified harmine that is able to induce ß-cell proliferation both in vitro and in vivo. Since harmine has suboptimal kinase selectivity, we sought to expand structure-activity relationships for harmine's DYRK1A activity, to enhance selectivity, while retaining human ß-cell proliferation capability. We carried out the optimization of the 1-position of harmine and synthesized 15 harmine analogues. Six compounds showed excellent DYRK1A inhibition with IC50 in the range of 49.5-264 nM. Two compounds, 2-2 and 2-8, exhibited excellent human ß-cell proliferation at doses of 3-30 µM, and compound 2-2 showed improved kinase selectivity as compared to harmine.

Development of a reliable automated screening system to identify small molecules and biologics that promote human ß-cell regeneration.

Numerous compounds stimulate rodent ß-cell proliferation; however, translating these findings to human ß-cells remains a challenge. To examine human ß-cell proliferation in response to such compounds, we developed a medium-throughput in vitro method of quantifying adult human ß-cell proliferation markers. This method is based on high-content imaging of dispersed islet cells seeded in 384-well plates and automated cell counting that identifies fluorescently labeled ß-cells with high specificity using both nuclear and cytoplasmic markers. ß-Cells from each donor were assessed for their function and ability to enter the cell cycle by cotransduction with adenoviruses encoding cell cycle regulators cdk6 and cyclin D3. Using this approach, we tested 12 previously identified mitogens, including neurotransmitters, hormones, growth factors, and molecules, involved in adenosine and Tgf-1ß signaling. Each compound was tested in a wide concentration range either in the presence of basal (5 mM) or high (11 mM) glucose. Treatment with the control compound harmine, a Dyrk1a inhibitor, led to a significant increase in Ki-67+ ß-cells, whereas treatment with other compounds had limited to no effect on human ß-cell proliferation. This new scalable approach reduces the time and effort required for sensitive and specific evaluation of human ß-cell proliferation, thus allowing for increased testing of candidate human ß-cell mitogens.

A human islet cell culture system for high-throughput screening.

A small-molecule inducer of beta-cell proliferation in human islets represents a potential regeneration strategy for treating type 1 diabetes. However, the lack of suitable human beta cell lines makes such a discovery a challenge. Here, we adapted an islet cell culture system to high-throughput screening to identify such small molecules. We prepared microtiter plates containing extracellular matrix from a human bladder carcinoma cell line. Dissociated human islets were seeded onto these plates, cultured for up to 7 days, and assessed for proliferation by simultaneous Ki67 and C-peptide immunofluorescence. Importantly, this environment preserved beta-cell physiological function, as measured by glucose-stimulated insulin secretion. Adenoviral overexpression of cdk-6 and cyclin D(1), known inducers of human beta cell proliferation, was used as a positive control in our assay. This induction was inhibited by cotreatment with rapamycin, an immunosuppressant often used in islet transplantation. We then performed a pilot screen of 1280 compounds, observing some phenotypic effects on cells. This high-throughput human islet cell culture method can be used to assess various aspects of beta-cell biology on a relatively large number of compounds.

A 7-day continuous infusion of PTH or PTHrP suppresses bone formation and uncouples bone turnover.

Human in vivo models of primary hyperparathyroidism (HPT), humoral hypercalcemia of malignancy (HHM), or lactational bone mobilization for more than 48 hours have not been described previously. We therefore developed 7-day continuous-infusion models using human parathyroid hormone(1-34) [hPTH(1-34)] and human parathyroid hormone-related protein(1-36) [hPTHrP(1-36)] in healthy human adult volunteers. Study subjects developed sustained mild increases in serum calcium (10.0 mg/dL), with marked suppression of endogenous PTH(1-84). The maximal tolerated infused doses over a 7-day period (2 and 4 pmol/kg/h for PTH and PTHrP, respectively) were far lower than in prior, briefer human studies (8 to 28 pmol/kg/h). In contrast to prior reports using higher PTH and PTHrP doses, both 1,25-dihydroxyvitamin D(3) [1,25(OH)(2) D(3) ] and tubular maximum for phosphorus (TmP/GFR) remained unaltered with these low doses despite achievement of hypercalcemia and hypercalciuria. As expected, bone resorption increased rapidly and reversed promptly with cessation of the infusion. However, in contrast to events in primary HPT, bone formation was suppressed by 30% to 40% for the 7 days of the infusions. With cessation of PTH and PTHrP infusion, bone-formation markers abruptly rebounded upward, confirming that bone formation is suppressed by continuous PTH or PTHrP infusion. These studies demonstrate that continuous exposure of the human skeleton to PTH or PTHrP in vivo recruits and activates the bone-resorption program but causes sustained arrest in the osteoblast maturation program. These events would most closely mimic and model events in HHM. Although not a perfect model for lactation, the increase in resorption and the rebound increase in formation with cessation of the infusions are reminiscent of the maternal skeletal calcium mobilization and reversal that occur following lactation. The findings also highlight similarities and differences between the model and HPT.

The calcemic response to continuous parathyroid hormone (PTH)(1-34) infusion in end-stage kidney disease varies according to bone turnover: a potential role for PTH(7-84).

CONTEXT: Factors contributing to PTH resistance in dialysis patients remain elusive. OBJECTIVES: The study assessed the skeletal and biochemical response to 46 h of PTH(1-34) infusion in dialysis patients. DESIGN: The study was a prospective, controlled assessment of response to PTH(1-34). SETTING: The study was performed at the University of California, Los Angeles, General Clinical Research Center. PARTICIPANTS: Nineteen dialysis patients and 17 healthy volunteers were studied. INTERVENTION: PTH(1-34) was infused at a rate of 8 pmol/kg x h for 46 h. Bone biopsy was performed in all dialysis patients. MAIN OUTCOME MEASURES: Serum calcium, phosphorus, 1,25-dihydroxyvitamin D, PTH (four separate assays), and FGF-23 were determined at baseline and h 7, 23, 35, and 46 of the infusion. RESULTS: Serum calcium levels rose in healthy volunteers (9.2 +/- 0.1 to 11.9 +/- 0.3 mg/dl; P < 0.01) and in dialysis patients with adynamic/normal bone turnover (9.0 +/- 0.3 to 10.7 +/- 0.7 mg/dl; P < 0.05) but did not change in dialysis patients with high bone turnover. Serum phosphorus levels declined in healthy volunteers (3.9 +/- 0.1 to 3.5 +/- 0.1 mg/dl; P < 0.05) but increased in all dialysis patients (6.7 +/- 0.4 to 8.0 +/- 0.3 mg/dl; P < 0.05). Full-length PTH(1-84) declined in all subjects; however, PTH(7-84) fragments declined only in healthy subjects and in dialysis patients with normal/adynamic bone but remained unchanged in dialysis patients with high bone turnover. CONCLUSIONS: The skeleton of dialysis patients with high bone turnover is resistant to the calcemic actions of PTH. PTH(7-84) may contribute to this phenomenon.

Safety and tolerability of subcutaneous PTHrP(1-36) in healthy human volunteers: a dose escalation study.

Parathyroid hormone-related protein (PTHrP) is an anabolic skeletal agent in mice, rats and humans. In previous studies, we have demonstrated that PTHrP can be administered to osteoporotic postmenopausal women at a dose of 6.56 microg/kg/day (or approximately 400 microg/day) for 3 months to yield a 4.7% increase in lumbar spine BMD. This regimen was free of hypercalcemia or adverse effects. Moreover, PTHrP appeared to stimulate bone formation selectively, without stimulating bone resorption. This efficacy in the absence of adverse effects, as well as the apparent "pure anabolic" action of PTHrP, prompted us to attempt to define the complete therapeutic window for PTHrP. In this study, we gradually escalated the dose of PTHrP(1-36) from 9 to 28 microg/kg (or approximately 570 microg to 1,946 microg) administered as a single subcutaneous dose to 22 healthy young adult subjects. PTHrP(1-36) was well tolerated even at the highest dose, just under 2.0 mg, some five times higher than we have previously demonstrated to be effective in increasing bone mass, and some 100 times higher than the maximal approved dose of PTH(1-34). Despite the large dose of PTHrP, the highest serum calcium achieved was 10.6 mg/dl, and this was observed in only one subject at the highest dose. The mean serum calcium in subjects receiving the highest dose was 9.6 mg/dl. Only one subject experienced adverse symptoms/signs, and this was at the highest dose. We conclude that subcutaneous PTHrP(1-36) is safe when administered in single doses approaching 2.0 mg. These findings indicate that the therapeutic window for PTHrP(1-36) in humans is wide and permit the design and implementation of longer safety and efficacy trials.

Direct comparison of sustained infusion of human parathyroid hormone-related protein-(1-36) [hPTHrP-(1-36)] versus hPTH-(1-34) on serum calcium, plasma 1,25-dihydroxyvitamin D concentrations, and fractional calcium excretion in healthy human volunteers.

PTH and PTH-related protein (PTHrP) cause primary hyperparathyroidism and humoral hypercalcemia of malignancy (HHM), respectively. These syndromes are similar in several important ways, but differ in several characteristic, yet unexplained, ways. Two of the unresolved questions in HHM and hyperparathyroidism involve renal physiology. 1) Why does renal proximal tubular production of 1,25-dihydroxyvitamin D [1,25-(OH)(2)D] differ between the two syndromes? 2) Do distal tubular calcium responses to PTH and PTHrP differ in the two syndromes? To address these questions, we compared the two peptides, human PTH-(1-34) and PTHrP-(1-36), in a direct, head to head study using a continuous, steady state infusion of each peptide at the same dose in normal human volunteers for 46 h. We had previously described such methods as applied to PTHrP, but a direct multiday comparison of PTHrP to PTH has not previously been reported. In two groups (seven subjects each) of healthy young (25- to 35-yr-old) normal volunteers, PTH and PTHrP infused at 8 pmol/kg.h displayed similar calcemic effects, although PTH was slightly more potent in this regard. Both peptides also displayed similar phosphaturic effects. In addition, both peptides had similar effects on renal tubular calcium handling, yielding fractional calcium excretion values of approximately 3.5%, some 50% below the values (6.5%) observed in subjects rendered similarly hypercalcemic by the infusion of calcium. In contrast to these several quantitatively similar effects of PTH and PTHrP, PTH tended to be selectively more effective than PTHrP in stimulating renal production of 1,25-(OH)(2)D. These studies indicate that renal tubular calcium reabsorption is likely to contribute to hypercalcemia in patients with HHM. In addition, PTH may be selectively more effective than PTHrP in stimulating 1,25-(OH)(2)D production, in contrast to its phosphaturic, calcemic effects and its effects to stimulate nephrogenous cAMP excretion and renal tubular calcium reabsorption.

Short-term, high-dose parathyroid hormone-related protein as a skeletal anabolic agent for the treatment of postmenopausal osteoporosis.

PTH-related protein (PTHrP) is homologous with PTH. PTH, an effective anabolic agent for treating osteoporosis, has been shown to stimulate both bone resorption by osteoclasts and bone formation by osteoblasts. We examined whether PTHrP might share anabolic properties in osteoporosis. A 3-month double-blind, prospective, placebo-controlled, randomized clinical trial was performed in 16 healthy postmenopausal women with osteoporosis. All received calcium and vitamin D, and all continued their prior hormone replacement therapy. One group also received daily sc PTHrP (6.56 microg/kg x d, or approximately 400 microg/d), and the other group received placebo injections. The PTHrP group displayed a 4.7% increase in lumbar spine bone mineral density (BMD) and also demonstrated an increase in osteoblastic bone formation, as assessed using serum osteocalcin measurements. In contrast, there was no increase in bone-specific alkaline phosphatase and collagen-1 propeptide or either of two markers of osteoclastic bone resorption, N-telopeptide, or deoxypyridinoline. One subject in the placebo group withdrew from the study, but there were no significant adverse events in the PTHrP group. PTHrP administered sc in high doses for only 3 months appears to be a potent anabolic agent, producing a 4.7% increase in lumbar spine BMD. This compares very favorably to available antiresorptive drugs for osteoporosis and is similar to the increases in BMD at this early time point reported for PTH. Despite the high doses, PTHrP was well tolerated. Larger clinical trials are required to confirm these results and fully assess the anabolic potential of PTHrP in osteoporosis.