Inhibition of Interleukin-33 to Reduce Glomerular Endothelial Inflammation in Diabetic Kidney Disease.

Introduction: Inflammation is a significant contributor to cardiorenal morbidity and mortality in diabetic kidney disease (DKD). The pathophysiological mechanisms linking systemic, subacute inflammation and local, kidney injury-initiated immune maladaptation is partially understood. Methods: Here, we explored the expression of proinflammatory cytokines in patients with DKD; investigated mouse models of type 1 and type 2 diabetes (T2D); evaluated glomerular signaling in vitro; performed post hoc analyses of systemic and urinary markers of inflammation; and initiated a phase 2b clinical study (FRONTIER-1; NCT04170543). Results: Transcriptomic profiling of kidney biopsies from patients with DKD revealed significant glomerular upregulation of interleukin-33 (IL-33). Inhibition of IL-33 signaling reduced glomerular damage and albuminuria in the uninephrectomized db/db mouse model (T2D/DKD). On a cellular level, inhibiting IL-33 improved glomerular endothelial health by decreasing cellular inflammation and reducing release of proinflammatory cytokines. Therefore, FRONTIER-1 was designed to test the safety and efficacy of the IL-33-targeted monoclonal antibody tozorakimab in patients with DKD. So far, 578 patients are enrolled in FRONTIER-1. The baseline inflammation status of participants (N > 146) was assessed in blood and urine. Comparison to independent reference cohorts (N > 200) validated the distribution of urinary tumor necrosis factor receptor 1 (TNFR1) and C-C motif chemokine ligand 2 (CCL2). Treatment with dapagliflozin for 6 weeks did not alter these biomarkers significantly. Conclusion: We show that blocking the IL-33 pathway may mitigate glomerular endothelial inflammation in DKD. The findings from the FRONTIER-1 study will provide valuable insights into the therapeutic potential of IL-33 inhibition in DKD.

Injectable Biodegradable Silica Depot: Two Months of Sustained Release of the Blood Glucose Lowering Peptide, Pramlintide.

Diabetes mellitus is a major healthcare challenge. Pramlintide, a peptide analogue of the hormone amylin, is currently used as an adjunct with insulin for patients who fail to achieve glycemic control with only insulin therapy. However, hypoglycemia is the dominant risk factor associated with such approaches and careful dosing of both drugs is needed. To mitigate this risk factor and compliance issues related to multiple dosing of different drugs, sustained delivery of Pramlintide from silica depot administered subcutaneously (SC) was investigated in a rat model. The pramlintide-silica microparticle hydrogel depot was formulated by spray drying of silica sol-gels. In vitro dissolution tests revealed an initial burst of pramlintide followed by controlled release due to the dissolution of the silica matrix. At higher dosing, pramlintide released from subcutaneously administered silica depot in rats showed a steady concentration of 500 pM in serum for 60 days. Released pramlintide retained its pharmacological activity in vivo, as evidenced by loss of weight. The biodegradable silica matrix offers a sustained release of pramlintide for at least two months in the rat model and shows potential for clinical applications.

Use of CRISPR/Cas9-engineered INS-1 pancreatic ß cells to define the pharmacology of dual GIPR/GLP-1R agonists.

Dual-agonist molecules combining glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) activity represent an exciting therapeutic strategy for diabetes treatment. Although challenging due to shared downstream signalling pathways, determining the relative activity of dual agonists at each receptor is essential when developing potential novel therapeutics. The challenge is exacerbated in physiologically relevant cell systems expressing both receptors. To this end, either GIP receptors (GIPR) or GLP-1 receptors (GLP-1R) were ablated via RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 endonucleases in the INS-1 pancreatic ß-cell line. Multiple clonal cell lines harbouring gene disruptions for each receptor were isolated and assayed for receptor activity to identify functional knockouts (KOs). cAMP production in response to GIPR or GLP-1R activation was abolished and GIP- or GLP-1-induced potentiation of glucose-stimulated insulin secretion (GSIS) was attenuated in the cognate KO cell lines. The contributions of individual receptors derived from cAMP and GSIS assays were confirmed in vivo using GLP-1R KO mice in combination with a monoclonal antibody antagonist of GIPR. We have successfully applied CRISPR/Cas9-engineered cell lines to determining selectivity and relative potency contributions of dual-agonist molecules targeting receptors with overlapping native expression profiles and downstream signalling pathways. Specifically, we have characterised molecules as biased towards GIPR or GLP-1R, or with relatively balanced potency in a physiologically relevant ß-cell system. This demonstrates the broad utility of CRISPR/Cas9 when applied to native expression systems for the development of drugs that target multiple receptors, particularly where the balance of receptor activity is critical.

Hypomorphism in human NSMCE2 linked to primordial dwarfism and insulin resistance.

Structural maintenance of chromosomes (SMC) complexes are essential for maintaining chromatin structure and regulating gene expression. Two the three known SMC complexes, cohesin and condensin, are important for sister chromatid cohesion and condensation, respectively; however, the function of the third complex, SMC5-6, which includes the E3 SUMO-ligase NSMCE2 (also widely known as MMS21) is less clear. Here, we characterized 2 patients with primordial dwarfism, extreme insulin resistance, and gonadal failure and identified compound heterozygous frameshift mutations in NSMCE2. Both mutations reduced NSMCE2 expression in patient cells. Primary cells from one patient showed increased micronucleus and nucleoplasmic bridge formation, delayed recovery of DNA synthesis, and reduced formation of foci containing Bloom syndrome helicase (BLM) after hydroxyurea-induced replication fork stalling. These nuclear abnormalities in patient dermal fibroblast were restored by expression of WT NSMCE2, but not a mutant form lacking SUMO-ligase activity. Furthermore, in zebrafish, knockdown of the NSMCE2 ortholog produced dwarfism, which was ameliorated by reexpression of WT, but not SUMO-ligase-deficient NSMCE. Collectively, these findings support a role for NSMCE2 in recovery from DNA damage and raise the possibility that loss of its function produces dwarfism through reduced tolerance of replicative stress.

Prox1 ablation in hepatic progenitors causes defective hepatocyte specification and increases biliary cell commitment.

The liver has multiple functions that preserve homeostasis. Liver diseases are debilitating, costly and often result in death. Elucidating the developmental mechanisms that establish the liver's architecture or generate the cellular diversity of this organ should help advance the prevention, diagnosis and treatment of hepatic diseases. We previously reported that migration of early hepatic precursors away from the gut epithelium requires the activity of the homeobox gene Prox1. Here, we show that Prox1 is a novel regulator of cell differentiation and morphogenesis during hepatogenesis. Prox1 ablation in bipotent hepatoblasts dramatically reduced the expression of multiple hepatocyte genes and led to very defective hepatocyte morphogenesis. As a result, abnormal epithelial structures expressing hepatocyte and cholangiocyte markers or resembling ectopic bile ducts developed in the Prox1-deficient liver parenchyma. By contrast, excessive commitment of hepatoblasts into cholangiocytes, premature intrahepatic bile duct morphogenesis, and biliary hyperplasia occurred in periportal areas of Prox1-deficient livers. Together, these abnormalities indicate that Prox1 activity is necessary to correctly allocate cell fates in liver precursors. These results increase our understanding of differentiation anomalies in pathological conditions and will contribute to improving stem cell protocols in which differentiation is directed towards hepatocytes and cholangiocytes.

The emerging use of zebrafish to model metabolic disease.

The zebrafish research community is celebrating! The zebrafish genome has recently been sequenced, the Zebrafish Mutation Project (launched by the Wellcome Trust Sanger Institute) has published the results of its first large-scale ethylnitrosourea (ENU) mutagenesis screen, and a host of new techniques, such as the genome editing technologies TALEN and CRISPR-Cas, are enabling specific mutations to be created in model organisms and investigated in vivo. The zebrafish truly seems to be coming of age. These powerful resources invoke the question of whether zebrafish can be increasingly used to model human disease, particularly common, chronic diseases of metabolism such as obesity and type 2 diabetes. In recent years, there has been considerable success, mainly from genomic approaches, in identifying genetic variants that are associated with these conditions in humans; however, mechanistic insights into the role of implicated disease loci are lacking. In this Review, we highlight some of the advantages and disadvantages of zebrafish to address the organism's utility as a model system for human metabolic diseases.

Role of RIP140 in metabolic tissues: connections to disease.

The control of physiological processes requires the regulation and coordination of many different signals and is determined in part by the activation and repression of expression of specific target genes. RIP140 is a ligand dependent coregulator of many nuclear receptors that influence such diverse processes as muscle metabolism, adipocyte and hepatocyte function, and reproduction. Recent evidence has shown that the ability of RIP140 to regulate nuclear receptor function is determined by the relative level of RIP140 expression in comparison with other cofactors, by post-translational modifications and by interactions with additional transcription factors. As a result it is becoming apparent that RIP140, via its interplay with other coregulators, plays a fundamental role in determining both the normal and pathogenic physiological state.

The transcriptional corepressor RIP140 regulates oxidative metabolism in skeletal muscle.

Nuclear receptor signaling plays an important role in energy metabolism. In this study we demonstrate that the nuclear receptor corepressor RIP140 is a key regulator of metabolism in skeletal muscle. RIP140 is expressed in a fiber type-specific manner, and manipulation of its levels in null, heterozygous, and transgenic mice demonstrate that low levels promote while increased expression suppresses the formation of oxidative fibers. Expression profiling reveals global changes in the expression of genes implicated in both myofiber phenotype and metabolic functions. Genes involved in fatty-acid oxidation, oxidative phosphorylation, and mitochondrial biogenesis are upregulated in the absence of RIP140. Analysis of cultured myofibers demonstrates that the changes in expression are intrinsic to muscle cells and that nuclear receptor-regulated genes are direct targets for repression by RIP140. Therefore RIP140 is an important signaling factor in the regulation of skeletal muscle function and physiology.

The nuclear receptor cofactor, receptor-interacting protein 140, is required for the regulation of hepatic lipid and glucose metabolism by liver X receptor.

The liver X receptors (LXRs) are nuclear receptors that play important roles in the regulation of lipid metabolism. In this study, we demonstrate that receptor-interacting protein 140 (RIP140) is a cofactor for LXR in liver. Analysis of RIP140 null mice and hepatocytes depleted of RIP140 indicate that the cofactor is essential for the ability of LXR to activate the expression of a set of genes required for lipogenesis. Furthermore we demonstrate that RIP140 is required for the ability of LXR to repress the expression of the phosphoenolpyruvate carboxykinase gene in Fao cells and mice. Thus, we conclude that the function of RIP140 as a cofactor for LXR in liver varies according to the target genes and metabolic process, serving as a coactivator in lipogenesis but as a corepressor in gluconeogenesis.

Receptor interacting protein 140 regulates expression of uncoupling protein 1 in adipocytes through specific peroxisome proliferator activated receptor isoforms and estrogen-related receptor alpha.

Expression of uncoupling protein 1 (Ucp1) mRNA is elevated in differentiated adipocytes derived from brown or white adipose tissue devoid of the nuclear receptor corepressor receptor interacting protein 140 (RIP140). Increased expression is mediated in part by the recruitment of peroxisome proliferator activated receptors alpha and gamma, together with estrogen-related receptor alpha, which functions through a novel binding site on the Ucp1 enhancer. This demonstrates that regulation of Ucp1 expression in the absence of RIP140 involves derepression of at least three different nuclear receptors. The ability to increase expression of Ucp1 by beta-adrenergic signaling is independent of RIP140, as shown by the action of the beta(3)-adrenergic agonist CL 316,243 to stimulate expression in both brown and white adipocytes in the presence and absence of the corepressor. Therefore, the expression of this metabolic uncoupling protein in adipose cells is regulated by inhibition as well as activation of distinct signaling pathways.

Neuromedin U partially mediates leptin-induced hypothalamo-pituitary adrenal (HPA) stimulation and has a physiological role in the regulation of the HPA axis in the rat.

Intracerebroventricular (ICV) administration of the hypothalamic neuropeptide neuromedin U (NMU) or the adipostat hormone leptin increases plasma ACTH and corticosterone. The relationship between leptin and NMU in the regulation of the hypothalamo-pituitary adrenal (HPA) axis is currently unknown. In this study, leptin (1 nm) significantly increased the release of CRH from ex vivo hypothalamic explants by 207 +/- 8.4% (P < 0.05 vs. basal), an effect blocked by the administration of anti-NMU IgG. The ICV administration of leptin (10 mug, 0.625 nmol) increased plasma ACTH and corticosterone 20 min after injection [plasma ACTH (picograms per milliliter): vehicle, 63 +/- 20, leptin, 135 +/- 36, P < 0.05; plasma corticosterone (nanograms per milliliter): vehicle, 285 +/- 39, leptin, 452 +/- 44, P < 0.01]. These effects were partially attenuated by the prior administration of anti-NMU IgG. Peripheral leptin also stimulated ACTH release, an effect attenuated by prior ICV administration of anti-NMU IgG. We examined the diurnal pattern of hypothalamic NMU mRNA expression and peptide content, plasma leptin, and plasma corticosterone. The diurnal changes in hypothalamic NMU mRNA expression were positively correlated with hypothalamic NMU peptide content, plasma corticosterone, and plasma leptin. The ICV administration of anti-NMU IgG significantly attenuated the dark phase rise in corticosterone [corticosterone (nanograms per milliliter): vehicle, 493 +/- 38; NMU IgG, 342 +/- 47 (P < 0.05)]. These studies suggest that NMU may play a role in the regulation of the HPA axis and partially mediate leptin-induced HPA stimulation.

Suppression of oxidative metabolism and mitochondrial biogenesis by the transcriptional corepressor RIP140 in mouse adipocytes.

Using an siRNA-based screen, we identified the transcriptional corepressor RIP140 as a negative regulator of insulin-responsive hexose uptake and oxidative metabolism in 3T3-L1 adipocytes. Affymetrix GeneChip profiling revealed that RIP140 depletion upregulates the expression of clusters of genes in the pathways of glucose uptake, glycolysis, TCA cycle, fatty acid oxidation, mitochondrial biogenesis, and oxidative phosphorylation in these cells. Conversely, we show that reexpression of RIP140 in mouse embryonic fibroblasts derived from RIP140-null mice downregulates expression of many of these same genes. Consistent with these microarray data, RIP140 gene silencing in cultured adipocytes increased both conversion of [14C]glucose to CO2 and mitochondrial oxygen consumption. RIP140-null mice, previously reported to resist weight gain on a high-fat diet, are shown here to display enhanced glucose tolerance and enhanced responsiveness to insulin compared with matched wild-type mice upon high-fat feeding. Mechanistically, RIP140 was found to require the nuclear receptor ERRalpha to regulate hexose uptake and mitochondrial proteins SDHB and CoxVb, although it likely acts through other nuclear receptors as well. We conclude that RIP140 is a major suppressor of adipocyte oxidative metabolism and mitochondrial biogenesis, as well as a negative regulator of whole-body glucose tolerance and energy expenditure in mice.

Neuromedin U has a physiological role in the regulation of food intake and partially mediates the effects of leptin.

Intracerebroventricular (ICV) administration of Neuromedin U (NMU), a hypothalamic neuropeptide, or leptin, an adipostat hormone released from adipose tissue, reduces food intake and increases energy expenditure. Leptin stimulates the release of NMU in vitro, and NMU expression is reduced in models of low or absent leptin. We investigated the role of NMU in mediating leptin-induced satiety. ICV administration of anti-NMU immunoglobulin G (IgG) (5 nmol) to satiated rats significantly increased food intake 4 h after injection, an effect seen for

Galanin-like peptide stimulates the release of gonadotropin-releasing hormone in vitro and may mediate the effects of leptin on the hypothalamo-pituitary-gonadal axis.

Leptin regulates the hypothalamo-pituitary-gonadal axis in relation to nutritional status. The mechanism through which leptin mediates its effects on neuroendocrine reproductive circuits remains unclear. Galanin-like peptide (GALP) is a recently identified hypothalamic peptide, localized in the arcuate nucleus, which seems to be regulated by leptin and stimulates LH when administered centrally. Here, we demonstrate that leptin stimulates the release of GALP and GnRH in vitro from hypothalamic explants harvested from male rats. In addition, we show that GALP stimulates the release of GnRH from hypothalamic explants and GT1-7 cells. Furthermore, we demonstrate that GALP antiserum blocks the stimulatory action of leptin on GnRH release from hypothalamic explants. GALP is a ligand of the galanin receptors. We therefore investigated whether the effect of GALP on GnRH release may be mediated via a known galanin receptor. GALP-stimulated GnRH release from hypothalamic explants was attenuated (but not abolished) by the galanin receptor antagonist galantide. However, GALP-stimulated GnRH release from GT1-7 cells was not diminished by the coadministration of galantide. In addition, none of the cloned galanin receptors were expressed in GT1-7 cells by RT-PCR. These observations suggest that GALP may stimulate GnRH release through an indirect pathway involving a galanin receptor and via a direct action on GnRH neurons, possibly through a novel receptor. These findings suggest that GALP may mediate the actions of leptin on the reproductive axis and provide a link between nutrition and fertility.

A role for arcuate cocaine and amphetamine-regulated transcript in hyperphagia, thermogenesis, and cold adaptation.

We have recently shown that injection of the hypothalamic peptide cocaine and amphetamine regulated transcript (CART) into discrete hypothalamic nuclei stimulates food intake. This stimulation was particularly marked in the arcuate nucleus. Here we show that twice daily intra-arcuate injection of 0.2 nmole CART peptide for 7 days was associated with a 60% higher daytime food intake, an 85% higher thermogenic response to the beta3 agonist BRL 35135, and a 60% increase in brown adipose tissue UCP-1 mRNA. In a separate study, using stereotactically targeted gene transfer, a CART transgene was delivered by using polyethylenimine to the arcuate nucleus of adult rats. Food intake was increased significantly during ad libitum feeding and following periods of food withdrawal and food restriction in CART over-expressing animals. CART over-expressing animals lost 12% more weight than controls following a 24-h fast. Brown adipose tissue uncoupling protein-1 (UCP-1) mRNA levels (collected Day 25) were 80% higher in CART over-expressing animals. Finally, by using quantitative in situ hybridization, we found that chronic cold exposure (20 days at 4oC) increased arcuate nucleus CART mRNA by 124%. Together with the orexigenic and thermogenic effects of CART, this finding suggests a role for arcuate nucleus CART in cold adaptation.

Effects of galanin-like peptide on food intake and the hypothalamo-pituitary-thyroid axis.

Galanin-like peptide (GALP) is a novel hypothalamic peptide synthesised in neurons in the arcuate nucleus which project to the paraventricular nucleus (PVN). GALP has recently been identified as an orexigenic peptide. In this study we aimed to further characterise the hypothalamic action of this peptide in energy homeostasis. Firstly, we investigated the orexigenic effect of GALP in the PVN and compared its effects with galanin and galanin 2-29. Secondly, we examined the effect of PVN administration of GALP and galanin on circulating thyroid-stimulating hormone (TSH). PVN administration of GALP significantly increased the food intake of satiated rats 1 h after administration at doses of 0.3, 1 and 3 nmol. In comparison with paraventricular administration of galanin, GALP was a more potent orexigen, whereas galanin 2-29, the relatively selective GAL R2 agonist, had no effect on food intake. Both GALP and galanin administration (1 nmol) into the PVN significantly decreased the level of circulating TSH. To investigate the mechanism of these effects, we examined the effect of GALP and galanin application on neuropeptide release from hypothalamic explants in vitro. GALP peptide (100 nM) stimulated the release of the orexigenic peptide neuropeptide Y from hypothalamic explants and decreased the release of the anorectic peptide cocaine-and-amphetamine-regulated transcript, whereas galanin (100 nM) peptide had no significant effect on the release of either peptide. Both GALP (100 nM) and galanin (100 nM) inhibited the release thyrotrophin-releasing hormone. These data suggest that in the PVN, GALP may play a role in energy homeostasis by stimulating food intake and suppressing TSH release.

Paraventricular nucleus administration of calcitonin gene-related peptide inhibits food intake and stimulates the hypothalamo-pituitary-adrenal axis.

Calcitonin gene-related protein (CGRP) inhibits food intake and stimulates the hypothalamo-pituitary-adrenal (HPA) axis after intracerebroventricular injection in rats. However, the hypothalamic site and mechanism of action are unknown. We investigated the effects of intraparaventricular nucleus administration (iPVN) of CGRP on food intake and the HPA axis in rats and the effect of CGRP on the release of hypothalamic neuropeptides in vitro. In addition, we investigated the effects of food deprivation on hypothalamic CGRP expression. CGRP dose-dependently reduced food intake in the first hour after iPVN injection in fasted male rats (saline, 5.1 +/- 0.8 g; 0.3 nmol CGRP, 1.1 +/- 0.5 g; P < 0.001 vs. saline). iPVN injection of CGRP(8-37) (a CGRP(1) receptor antagonist) alone had no effect on food intake. However, the reduction in food intake by iPVN CGRP was attenuated by prior administration of CGRP(8-37) [CGRP(8-37) (10 nmol)/CGRP (0.3 nmol), 3.0 +/- 0.8 g; P < 0.05 vs. 0.3 nmol CGRP]. CGRP (100 nM) stimulated the release of alpha-melanocyte stimulating hormone, cocaine- and amphetamine-related transcript, corticotropin-releasing hormone, and arginine vasopressin from hypothalamic explants to 127 +/- 19%, 148 +/- 10%, 158 +/- 17%, and 198 +/- 21% of basal levels, respectively (P < 0.05 vs. basal), but did not alter the release of either neuropeptide Y or agouti-related protein. Hypothalamic CGRP mRNA levels in 24-h fasted rats were increased to 130 +/- 8% of control levels [CGRP mRNA (arbitrary units), 4.75 +/- 0.4; controls, 3.65 +/- 0.34; P < 0.05]. Our data suggest that CGRP administered to the PVN inhibits food intake and stimulates the HPA axis.

Repeated ICV administration of oxyntomodulin causes a greater reduction in body weight gain than in pair-fed rats.

Oxyntomodulin (OXM) is a product of proglucagon processing in the intestine and the central nervous system. We reported that intracerebroventricular (ICV) and intranuclear administration of OXM caused an inhibition of food intake in rats (Dakin CL, Gunn I, Small CJ, Edwards CM, Hay DL, Smith DM, Ghatei MA, and Bloom SR. Endocrinology 142: 4244-4250, 2001). In this study, we investigated the effect of twice-daily ICV administration of OXM, 1 nmol, for 7 days. A pair-fed control was included. These animals were restricted to the food intake of the OXM group but injected twice daily with saline. OXM-treated animals gained significantly less weight than either control group (day 8: OXM, 12.2 +/- 1.9 g vs. pair fed, 21.0 +/- 2.1 g; P < 0.005). OXM treatment caused a reduction in epididymal white adipose tissue (OXM, 1.13 +/- 0.03 g vs. pair fed, 1.29 +/- 0.04 g; P < 0.05) and interscapular brown adipose tissue (OXM, 0.15 +/- 0.01 g vs. pair fed, 0.18 +/- 0.01 g; P < 0.05) and increased core temperature compared with saline control, suggestive of enhanced energy expenditure. The food restriction-induced suppression in plasma TSH, seen in the pair-fed group, was prevented by OXM, potentially via increased release of hypothalamic TRH. In summary, ICV OXM causes reduced body weight gain and body adiposity following chronic administration.