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Modified UCN2 Peptide Acts As an Insulin Sensitizer in Skeletal Muscle

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Modified UCN2 Peptide Acts As an Insulin Sensitizer in Skeletal Muscle Diabetes Volume 68, July 2019 1403 Modified UCN2 Peptide Acts as an Insulin Sensitizer in Skeletal Muscle of Obese Mice Melissa L. Borg,1 Julie Massart,2 Milena Schönke,2 Thais De Castro Barbosa,1 Lili Guo,3 Mark Wade,3 Jorge Alsina-Fernandez,3 Rebecca Miles,3 Andrew Ryan,3 Steve Bauer,3 Tamer Coskun,3 Elizabeth O’Farrell,3 Evan M. Niemeier,3 Alexander V. Chibalin,2 Anna Krook,1 Håkan K. Karlsson,2 Joseph T. Brozinick,3 and Juleen R. Zierath1,2 Diabetes 2019;68:1403–1414 | https://doi.org/10.2337/db18-1237 The neuropeptide urocortin 2 (UCN2) and its receptor enhances skeletal muscle insulin sensitivity and mitochon- corticotropin-releasing hormone receptor 2 (CRHR2) are drial function (1). Nevertheless, such lifestyle interven- highly expressed in skeletal muscle and play a role in tions have poor adherence, requiring pharmacological regulating energy balance and glucose metabolism. advances to alleviate obesity and prevent metabolic dis- We investigated a modified UCN2 peptide as a poten- ease. Consequently, efforts are under way to develop in- tial therapeutic agent for the treatment of obesity and sulin sensitizers and weight-reducing pharmacological fi METABOLISM insulin resistance, with a speci c focus on skeletal agents for the treatment of diabetes (2,3). – muscle. High-fat fed mice (C57BL/6J) were injected Skeletal muscle is an important tissue involved in daily with a PEGylated UCN2 peptide (compound A) at maintaining glucose homeostasis under insulin-stimulated 0.3 mg/kg subcutaneously for 14 days. Compound A conditions and is a major site of insulin resistance in type reduced body weight, food intake, whole-body fat mass, 2 diabetes (4,5). Although precise mechanisms of skeletal and intramuscular triglycerides compared with vehicle- muscle insulin resistance are not fully elucidated, impaired treated controls. Furthermore, whole-body glucose tol- erance was improved by compound A treatment, with insulin signaling and reduced glucose uptake are major increased insulin-stimulated Akt phosphorylation at aspects (4,5). Insulin resistance is present at all pathogenic Ser473 and Thr308 in skeletal muscle, concomitant with stages of type 2 diabetes progression. Consequently, increased glucose transport into extensor digitorum lon- efforts to maintain skeletal muscle insulin sensitivity to gus and gastrocnemius muscle. Mechanistically, this is prevent/delay type 2 diabetes are warranted. In addition linked to a direct effect on skeletal muscle because to lifestyle modifications, including diet and exercise, new ex vivo exposure of soleus muscle from chow-fed lean therapeutic routes to directly enhance skeletal muscle mice to compound A increased glucose transport and insulin sensitivity, either as monotherapy or in combi- insulin signaling. Moreover, exposure of GLUT4-Myc– nation with other drugs, are of interest to treat type 2 labeled L6 myoblasts to compound A increased GLUT4 diabetes. trafficking. Our results demonstrate that modified UCN2 The corticotropin-releasing factor (CRF) urocortin peptides may be efficacious in the treatment of type 2 (UCN) family of neuropeptides is a direct modulator of diabetes by acting as an insulin sensitizer in skeletal muscle. the hypothalamic-pituitary-adrenal axis both centrally and peripherally (6). Within this family are four peptides (CRF andUCN1,2,and3)thatarestructurallyrelatedbut Exercise and diet are potent lifestyle interventions to encoded by separate genes (7). UCN peptides signal combat metabolic dysfunction by improving weight man- through two different G-protein–coupled receptors: agement and glucose homeostasis. In particular, exercise corticotropin-releasing hormone receptors (CRHRs) 1 1Department of Physiology and Pharmacology, Section for Integrative Physiology, This article contains Supplementary Data online at http://diabetes Karolinska Institutet, Stockholm, Sweden .diabetesjournals.org/lookup/suppl/doi:10.2337/db18-1237/-/DC1. 2 Department of Molecular Medicine and Surgery, Section for Integrative Physi- © 2019 by the American Diabetes Association. Readers may use this article as ology, Karolinska Institutet, Stockholm, Sweden longastheworkisproperlycited,theuseiseducationalandnotforprofit, and the work 3 Lilly Research Laboratories, Division of Eli Lilly and Company, Indianapolis, IN is not altered. More information is available at http://www.diabetesjournals.org/ Corresponding author: Juleen R. Zierath, [email protected] content/license. Received 21 November 2018 and accepted 8 April 2019 1404 UCN2 and Skeletal Muscle Insulin Sensitivity Diabetes Volume 68, July 2019 and 2 (8). These peptides and receptors are differentially liquid chromatography with tandem mass spectrometry. expressed in central and peripheral tissues (7,8). UCN1 Pharmacokinetic parameters were calculated by noncom- binds to both receptors, while UCN2 and UCN3 are partmental analysis using Phoenix WinNonlin 6.3 software. selective for CRHR2. Skeletal muscle has high expression levels of both UCN2 and its receptor CRHR2 (9). While cAMP Assay emerging evidence suggests that CRF peptides regulate HEK293 cells transfected with mouse CRHR1 or CRHR2b cardiovascular and renal function and inflammatory pro- plasmid were plated in 96-well plates at 2,000 cells per well cesses (10), their role in metabolic diseases is unclear. and allowed to attach overnight. Serial dilutions of hu- UCNs and CRHR2 play a role in glucose homeostasis. man UCN2 or compound A were placed onto the cells Ucn2 and Crhr2 knockout mice have enhanced glucose for 15 min. cAMP levels were measured using a cAMP tolerance and increased insulin sensitivity and are pro- cell-based assay kit (Cisbio). tected from high-fat diet (HFD)–induced obesity (11,12). HFD and/or elevated stress states upregulate skeletal L6-GLUT4-Myc Cell Surface Detection muscle CRHR2 (13), while CRHR2 activation inhibits L6 rat myoblasts expressing human GLUT4 with an exo- insulin signaling (14). Thus, increased CRHR2 activity facial Myc-epitope tag were cultured in a 96-well plate and impairs glucose homeostasis. In contrast, whole-body incubated in the absence or presence of 100 nmol/L in- Ucn3 transgenic mice are protected from HFD-induced sulin, 100 nmol/L compound A, or 100 nmol/L clenbuterol obesity (15), and transient overexpression of Ucn3 in for 30 min. Cell surface density of GLUT4-Myc was mea- skeletal muscle enhances glucose metabolism through sured as previously described (22). Fluorescence intensity increased insulin signaling (16). In addition, Ucn2 over- was obtained using a LI-COR Odyssey eXL (LI-COR Bio- expression through systemic virus delivery improves sciences, Lincoln, NE). whole-body insulin sensitivity in HFD-fed rodents (17). Accordingly, activating CRHR2 during obesity can also Animals enhance glucose homeostasis. While the UCN-CRHR axis Experiments were approved by the Stockholm North an- appears to regulate skeletal muscle metabolism, the pre- imal ethics committee or the Eli Lilly institutional animal dominant effects remain unclear. care and use committee. Male mice (C57BL/6J) were Observations of aerobic training–like phenotypes in purchased from Charles River Laboratories (Sulzfeld, Ger- transgenic mice (18–20) has ignited interest in devel- many) or Envigo (Somerset, NJ) at 5 weeks of age. Mice oping pharmacological therapies to combat insulin re- were maintained under a 12-h light/dark cycle and had free sistance in patients with type 2 diabetes (21). Given the access to water and standard rodent chow (4% kcal from role of the UCN-CRHR axis in skeletal muscle metabo- fat, R34; Lantmännen, Kimstad, Sweden). At 6 weeks of lism, we hypothesized that UCN peptides act as insulin age, mice were placed on either a standard rodent chow or sensitizers in skeletal muscle. Thus, we investigated the an HFD (60% kcal from fat, TD.06414; Harlan Laborato- effects of a modified UCN2 peptide acting on the CHRH2 ries) ad libitum for 20 weeks and were single housed after in HFD-induced obese mice, with a specific focus on 19 weeks. After 20 weeks on an HFD, mice received daily skeletal muscle. subcutaneous injections of vehicle before the onset of the dark period (0.5% pan-albumin/0.9% NaCl) or compound A (0.3 mg/kg body weight) for 14 days. Injections were RESEARCH DESIGN AND METHODS performed in the intrascapular region or hind leg on Peptide Synthesis alternating days to minimize discomfort. Compound A (a PEGylated peptide analog of human UCN2) was synthesized using established solid-phase pep- Free Wheel Running fi tide synthesis protocols. After nal cleavage of the peptide HFD-fed mice were randomized into sedentary or wheel fi from the resin, the peptide was puri ed using reversed- running groups. Wheel running mice were acclimatized to phase chromatography and lyophilized to obtain peptide the running wheels for 7 days, and all groups were weight fl powder as tri uoroacetate salt. The peptide was conju- and running matched before injections. Body weight and gated to a 20-kDa functionalized polyethylene glycol (PEG) food intake were recorded daily. Activity of the mice on the polymer through an acetamide-based linker. Formulated running wheels (35-cm diameter) was monitored by a mag- aliquots of the peptide conjugate in PBS were stored netic switch affixed to each wheel, which recorded the 2 at 20°C. Working solutions were freshly prepared from number of revolutions. Data were captured by an automated thawed stock aliquots diluted with 0.5% pan-albumin/0.9% computer monitoring system (VitalView application soft- NaCl. ware; Mini-Mitter Company).
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