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). Physical activity was recorded continuously as wheel revolutions per 5-min interval. Pharmacokinetics Pharmacokinetics were determined in mice after a single Ex Vivo Glucose Uptake subcutaneous administration of compound A. Plasma con- Extensor digitorum longus (EDL) muscles were dissected centrations of compound A were determined through from 4-h–fasted mice anesthetized with an intraperitoneal diabetes.diabetesjournals.org Borg and Associates 1405 injection of 16 mL/g body weight 2.5% 2,2,2-tribromoethanol (BTX). Four days after electroporation, mice were sub- and tertiary amyl alcohol. Muscles were incubated with jected to in vivo glucose uptake. Krebs-Henseleit buffer under continuous gassing (95% O2/ Biochemical Analysis 5% CO2) at 30°C in the absence (basal) or presence of 0.36 nmol/L insulin (Actrapid; Novo Nordisk), and Glycogen and triacylglyceride (TAG) content in liver and – 2-deoxy-D-glucose uptake was determined as previ- TA muscle were measured in 4-h fasted mice using a Gly- ously described (23). Results are expressed as mmol/L cogen Assay Kit (ab65620; Abcam) or Triglyceride Quan- 2 2 glucose 3 mg protein 1 3 20 min 1. tification Assay Kit (ab65336; Abcam) according to the Soleus muscles from 8-week-old, chow-fed mice were manufacturer’s protocol. Plasma free fatty acid (ab65341; incubated in the absence or presence of compound A Abcam) and plasma leptin (MOB00; R&D Systems) were (63.3 nmol/L) with or without a submaximal insulin dose analyzed using assay kits according to the manufacturers’ (0.18 nmol/L) to assess insulin sensitivity. Glucose uptake protocol. was determined as described above. Western Blot Analysis In Vivo Glucose Uptake Western blot analysis was performed as previously de- Mice fed an HFD for 20 weeks received daily subcutaneous scribed (25). Primary antibodies used are listed in Supple- fi injections of compound A (0.3 mg/kg) or vehicle for 6 days. mentary Table 1. Bands were quanti ed using Quantity Fasted mice (4 h) were anesthetized with isoflurane. Mice One 1-D analysis software (Bio-Rad) and normalized to 3 total protein staining with Ponceau S (Sigma-Aldrich). received 10 mCi [ H]2-deoxy-D-glucose (PerkinElmer) 6 0.5 units/kg insulin (Humilin R; Eli Lilly) by retro-orbital Statistics injection. Blood samples were taken at 2, 5, 10, 15, 20, and Significant differences were determined by one-way, two- 30 min after injection, treated with Ba(OH) , and pre- 2 way, or two-way repeated-measures ANOVA with Sidak cipitated with ZnSO for determination of blood-specific 4 multiple comparison post hoc test as indicated in the figure activity. After centrifugation, the supernatant was col- legends. Chow-fed mice were excluded from statistical lected, and radioactivity was determined using liquid analysis because they served as a control for the HFD. scintillation counting (Beckman LSC). Animals were eu- Comparisons were considered significant at P , 0.05. thanized, and tissues were frozen. Tissue homogenates Analyses were performed using GraphPad 7 statistical were mixed with either water to determine total 2-deoxy- software (GraphPad Software). D-glucose or Ba(OH)2/ZnSO4 to determine unphosphory- lated 2-deoxy-D-glucose as previously described (24). RESULTS Characteristics of the Modified UCN2 Peptide Glucose Tolerance and Body Composition The modified human UCN2 peptide (compound A) is based Glucose tolerance and body composition were determined on day 11 of the treatment. Glucose (2 g/kg body weight) on the previously reported compound 8 (26). In contrast to compound 8, compound A includes a cysteine residue at was administered by intraperitoneal injection in 4-h– position 29, where a PEG 20,000 is attached through an fasted mice. Blood was sampled through the tail vein to acetamide-based linker. The potency of compound A was assess glucose (OneTouch Ultra 2 glucose meter; LifeScan) assessed by cAMP production in HEK293 cells transfected and insulin (Insulin ELISA Kit; Crystal Chem). Total lean with mouse CRHR1 or CRHR2b plasmid (the predominant and fat mass was assessed in conscious mice using the skeletal muscle isoform) (Table 1). CRHR2b-transfected EchoMRI-100 system (Echo Medical Systems). cells treated with serial dilutions of compound A for Electroporation Study 15 min had a half-maximal effective concentration value of 0.31 nmol/L compared with 0.08 nmol/L for human Chow-fed male mice (7–9 weeks of age) were anesthetized UCN2, while there was no cAMP production in cells trans- with isoflurane and a solution of 100 mL of hyaluronidase fected with CRHR1. Time to maximum plasma concentra- (Sigma H-3506) (2 mg/mL in Tyrode’s buffer) was injected tion after mice were treated with a single subcutaneous into the triceps surae and tibialis anterior (TA) (two injection of compound A was ;4 h, while the clearance was separate injections) in each leg 1 h before the DNA in- jection. Mice were then injected intramuscularly in the tibialis and triceps surae with 100 mghumanUCN2 — plasmid construct (catalog number RC201333, RefSeq Table 1 EC50 of response elicited by peptides b NM_033199.3; Origene) (two separate injections; 1 mg/mL Peptide CRHR1 EC50 CRHR2 EC50 in Tris-EDTA [TE] buffer) and an equal amount of TE Human UCN2 .10,000 (no activity) 0.08 6 0.03 buffer in the contralateral leg as a control. Thereafter, the Compound A .10,000 (no activity) 0.31 6 0.01 leg was subjected to electroporation (mode LV, 99 ms/500 V, Data are mean nmol/L 6 SD of compound added. The biological voltage 150 V, four 20-ms pulses one per second, activity of human UCN2 or compound A was assessed by 150 V/cm) using a BTX 830M electroporation unit determining cAMP production to serial dilutions. EC50, half-maximal effective concentration. (BTX, Holliston, MA) fitted with gene caliper electrodes 1406 UCN2 and Skeletal Muscle Insulin Sensitivity Diabetes Volume 68, July 2019 estimated at 5.94 mL/h/kg. Compound A has a half-life had reduced fasting blood glucose (Fig. 2A) and fasting of 22.3 h compared with 15 min for the native UCN2 plasma insulin (Fig. 2B) compared with HFD-fed vehicle- peptide (27). treated mice. Despite reduced plasma insulin (Fig. 2C), compound A–treated mice cleared the same amount of Chronic Activation of CRHR2 With Modified UCN2 blood glucose as vehicle-treated control mice during a glu- Reverses HFD-Induced Obesity cose tolerance test (GTT) (Fig. 2D and E). Moreover, in vivo To investigate the metabolic effects of modified UCN2 insulin-stimulated glucose uptake into red and white peptide, male mice were fed an HFD for 20 weeks before quadriceps and EDL from compound A–treated mice daily subcutaneous injections of vehicle or compound A was enhanced compared with insulin-stimulated vehicle- (0.3 mg/kg) for 14 days. Body weight was reduced in treated control muscles, while there was no alteration in compound A–treated mice after day 1, with a cumulative insulin-stimulated glucose uptake in brown adipose tissue weight loss of 7.5 g over the 14-day treatment period (Fig. (BAT) (Fig. 2F). 1A and B and Table 2). Weight loss in compound A–treated Although wheel running in vehicle-treated mice did not mice was accompanied by an 84% initial reduction in food affect fasting blood glucose levels compared with seden- intake compared with controls, which was followed by tary vehicle-treated mice (Fig. 2A), it reduced fasting a 28% decrease at the end of the treatment period (Fig. 1C plasma insulin levels (Fig. 2B). Wheel running vehicle- and Table 2). Thus, we noted a large decrease in food intake treated mice cleared the same amount of glucose during in the first several days and a smaller, but still significant a GTT as sedentary vehicle-treated mice (Fig. 2D and E), decrease later in the treatment. The reduction in food even with reduced plasma insulin (Fig. 2C). These data intake after the first injection with compound A is likely suggest that wheel running improves insulin sensitivity in due to an initial reduction in gastric emptying whereby HFD-fed mice. a single compound A injection reduced gastric emptying by Wheel running in compound A–treated mice reduced 52% compared with vehicle (Supplementary Fig. 1). Feed fasting blood glucose (Fig. 2A) and fasting plasma insulin efficiency (the ratio of body weight change to food intake) (Fig. 2B) compared with vehicle-treated wheel running was initially reduced 34% with compound A treatment, mice. Blood glucose clearance was increased in compound which was followed by a significant reduction at the end of A–treated wheel running mice compared with wheel run- the treatment period (Fig. 1D). Weight loss in compound ning controls (Fig. 2D and E), although similar plasma A–treated mice was attributed to a decrease in fat mass insulin levels were observed during the GTT (Fig. 2C). We without alteration in lean mass (Fig. 1E and Table 2). found no differences between compound A–treated sed- Compound A treatment reduced TAG content in TA muscle entary and compound A–treated wheel running mice in the compared with vehicle-treated mice (Fig. 1F). Compound A aforementioned parameters of in vivo glucose homeosta- treatment reduced liver weight without altering hepatic sis. Thus, while wheel running in vehicle-treated mice TAG or glycogen content (Table 1). improved glucose homeostasis, compound A treatment To investigate whether compound A treatment has alone was more potent, supporting the notion that a synergistic effect with physical activity, mice were given UCN2 peptide treatment in HFD-fed mice improves skel- free access to running wheels over the 14-day treatment etal muscle and whole-body insulin sensitivity with in- period. In vehicle-treated mice, wheel running reduced creased glucose uptake. body fat mass (Fig. 1E) without altering lean mass (Table 2), and this was associated with reduced final body weight Chronic Activation of CRHR2 With Modified UCN2 compared with sedentary mice (Table 2). Wheel running Improves Skeletal Muscle Insulin Sensitivity reduced the absolute and percent weight loss (Fig. 1A and To further investigate potential mechanisms of action for B)andfinal body weight in compound A–treated mice the positive effect of compound A on whole-body glucose compared with vehicle-treated wheel running mice (Table homeostasis, we assessed insulin sensitivity of EDL muscle 2), despite less distance ran (Supplementary Fig. 2). The ex vivo after compound treatment. Insulin-stimulated Akt weight loss was attributed to decreased fat mass (Fig. 1E) phosphorylation at Ser473 and Thr308 was increased in EDL without altering lean mass, liver weight (Table 2), or from compound A–treated mice compared with vehicle- TA TAGs (Fig. 1F) compared with vehicle-treated wheel treated mice (Fig. 3A and B). TBC1D4 phosphorylation at running mice. There were no synergistic effects with wheel Ser318 was increased above basal levels in compound A– running in the phenotypic improvements seen with treated EDL muscle (Supplementary Fig. 3A), whereas compound A treatment alone. This highlights the potent insulin-stimulated phosphorylation at Thr642 was de- nature of compound A on weight loss and fat mass creased compared with controls (Supplementary Fig. reduction. 3B). A trend for increased insulin-stimulated GSK3a phos- phorylation at Ser21 was observed in EDL muscle from Chronic Activation of CRHR2 With Modified UCN2 compound A–treated mice compared with vehicle-treated Improves In Vivo Glucose Homeostasis EDL (P = 0.087) (Supplementary Fig. 3C). Total Akt protein Next, we investigated the whole-body glucose homeostasis was increased in EDL muscle from compound A–treated of compound A–treated mice. Compound A–treated mice mice (Fig. 3D), whereas protein abundance of downstream diabetes.diabetesjournals.org Borg and Associates 1407

Figure 1—CRHR2 agonist reduces body weight (BW), food intake, and fat mass in HFD-fed mice. The following groups were studied: vehicle sedentary (Veh) (n = 9), vehicle wheel running (Veh Wheel) (n = 10), compound A sedentary (Comp A) (n = 10), and compound A wheel running (Comp A Wheel) (n = 10), all on an HFD. A chow-fed, sedentary, vehicle-treated group (Chow Veh) (n = 10) was included as a control. A and B: BW expressed as absolute and percent change from the first day of subcutaneous injections in sedentary mice and mice exposed to voluntary wheel running for the duration of the treatment. C and D: Food intake and feed efficiency (calculated by the ratio between BW loss and food intake) per day. Data are mean 6 SEM. ‡P , 0.05 main effect for UCN2 treatment; uP , 0.05 main effect for day; #P , 0.05 interaction; *Comp A vs. Veh; ^Comp A Wheel vs. Veh Wheel. E: MRI was performed on day 11 of treatment to measure total fat mass. F: TAG content of the TA. Dotted line indicates the mean of Chow Veh mice. Data are mean 6 SEM. ‡P , 0.05 main effect for UCN2 treatment; †P , 0.05 main effect for wheel running; *compared with vehicle of same condition; ¤compared with corresponding sedentary of same treatment as assessed by two-way ANOVA with Sidak post hoc analysis. BW and food intake assessed by two-way repeated-measures ANOVA with Sidak post hoc analysis. Sed, sedentary. 1408 UCN2 and Skeletal Muscle Insulin Sensitivity Diabetes Volume 68, July 2019

Table 2—Phenotypic characteristics of mice over 14-day treatment period HFD vehicle HFD compound A Chow vehicle Sedentary Wheel running Sedentary Wheel running Final body weight (g)‡† 31.9 6 0.6 47.9 6 1.2 43.8 6 0.9¤ 40.8 6 0.5* 39.4 6 0.9* Change in body weight (g)‡ 0.1 6 0.3 21.2 6 0.4 21.7 6 0.5 27.6 6 0.8* 26.4 6 0.5* Total food intake (kcal)‡ 167.4 6 6.6 184.0 6 8.4 184.4 6 5.9 115.4 6 7.5* 140.2 6 7.0*¤ Lean mass (g) 27.2 6 0.5 28.3 6 0.7 27.7 6 0.4 29.0 6 0.5 29.1 6 0.5 Plasma free fatty acids (nmol/mL) 0.4 6 0.1 0.3 6 0.0 0.3 6 0.0 0.3 6 0.0 0.3 6 0.0 Plasma leptin (ng/mL)‡ 5.8 6 1.3 99.4 6 7.4 87.5 6 9.4 27.2 6 4.5* 32.7 6 6.1* Liver weight (g)‡ 1.4 6 0.0 2.0 6 0.2 1.7 6 0.1 1.3 6 0.0* 1.4 6 0.1 Liver glycogen (mg/g tissue)† 230.8 6 27.7 142.3 6 22.0 185.3 6 17.4 121.1 6 18.2 174.0 6 28.6 Liver TAG (nmol/mg tissue) 8.9 6 0.8 19.8 6 6.3 12.2 6 2.2 10.8 6 1.5 13.2 6 2.1 Data are mean 6 SEM for n =8–10 mice per group. Phenotypic characteristics of vehicle- or compound A–treated mice over the 14-day treatment period. Mice were housed individually in cages without (sedentary) or with wheel running. ‡P , 0.05. Main effect for UCN2 treatment. †P , 0.05. Main effect for wheel running. *Compared with vehicle of same condition. ¤Compared with corresponding sedentary of same treatment as assessed by two-way ANOVA with Sidak post hoc analysis.

targets, such as GLUT 4, GSK3a,GSK3b,orglycogen (mTOR) phosphorylation at Ser2448 and Ser2481 was in- synthase, were unaltered (Supplementary Fig. 3D–F). creased in response to compound A in an insulin- Insulin-stimulated glucose transport into the EDL muscle independent manner (Fig. 4D, G, and H). was increased in compound A–treated mice compared with vehicle-treated mice (Fig. 3C). DISCUSSION Acute Activation of CRHR2 With Modified UCN2 Directly Enhances GLUT4 Translocation in L6 In the context of changing demographic patterns and the Myoblasts and Insulin Sensitivity in Soleus aging population, current pharmacological treatments to Skeletal Muscle combat the majority of lifestyle-related conditions are To assess the potential direct effect of UCN2 on skeletal inadequate. In particular, pharmacological treatments muscle metabolism, without the confounding factors of for type 2 diabetes that specifically target skeletal muscle reduced adiposity and food intake with in vivo compound to increase insulin sensitivity and preserve skeletal muscle A treatment, TA and triceps surae muscles from lean chow- function are lacking. In this regard, CRHR2 agonists may fed mice were electroporated with vectors containing improve skeletal muscle substrate metabolism and miti- human UCN2 or TE buffer in the contralateral leg. Ex- gate aging-associated disorders. Here, we determined pression of human UCN2 was detected in TA and soleus the effects of a modified UCN2 peptide in HFD, obese muscle after transfection with human UCN2 vectors (Sup- mice. We show that compound A treatment of HFD-fed plementary Fig. 4A). UCN2 overexpression in mouse mus- mice results in an initial reduction in food intake and rapid cle was associated with a modest reduction in endogenous weight loss, which was accompanied by improved whole- Ucn2 expression (Supplementary Fig. 4A). UCN2 overex- body glucose tolerance and insulin-stimulated glucose pression increased glucose transport into soleus muscle uptake into skeletal muscle. Mechanistically, this could compared with the control contralateral leg (Supplemen- be due to an effect on skeletal muscle because ex vivo tary Fig. 4B), confirming a positive regulation of skeletal stimulation of soleus muscle from lean chow-fed mice with muscle glucose transport by UCN2. In L6-GLUT4-Myc compound A increased glucose uptake and insulin signal- myoblasts, compound A (100 nmol/L) enhanced GLUT4 ing. Thus, UCN2 peptides may be efficacious in the treat- translocation to the membrane to levels comparable to ment of type 2 diabetes by acting as insulin sensitizers. insulin stimulation (100 nmol/L) (Fig. 4A and B). Com- Genetic manipulation of CRF family members alters pound A stimulation also increased glucose uptake ex vivo body weight in mouse models. While body weight in Ucn2 in skeletal muscle. Isolated soleus muscle from lean chow- knockout mice is unaltered after 16 weeks on an HFD, fat fed mice was incubated with compound A (63.3 nmol/L), mass is reduced, and lean mass is increased (11). Con- with or without submaximal insulin (0.18 nmol/L) for 1 h. versely, overexpression of Ucn3, which also signals through Compound A increased insulin-stimulated glucose uptake CRHR2, increases body weight, with increased lean mass into soleus muscle compared with insulin-stimulated ve- in chow-fed transgenic mice, whereas the HFD-fed trans- hicle treatment (Fig. 4C). Akt phosphorylation at both genic mice are obesity resistant (15). These genetic models Ser473 and Thr308 was increased in response to insulin and are at the whole-body level, and therefore, the contribution compound A stimulation compared with insulin stimu- of a centrally mediated effect on metabolism cannot be lation alone (Fig. 4C–E). Mammalian target of rapamycin excluded. Here, we provide evidence that pharmacological diabetes.diabetesjournals.org Borg and Associates 1409

Figure 2—CRHR2 agonist improves glucose homeostasis in HFD-fed mice. A and B: Fasting blood glucose and fasting plasma insulin. An intraperitoneal GTT was performed with 2 g/kg glucose. C and D: Plasma insulin at 15-min GTT and the incremental area under the curve (iAUC) during the GTT. The dotted line indicates the mean of chow vehicle (Veh) mice. Data are mean 6 SEM for n =9–10 mice per group. ‡P , 0.05 main effect for UCN2 treatment; #P , 0.05 interaction; *compared with Veh of same condition; ¤compared with corresponding sedentary (Sed) of same treatment as assessed by two-way ANOVA with Sidak post hoc analysis. E: Blood glucose during the GTT. ‡P , 0.05 main effect for UCN2 treatment; uP , 0.05 main effect for time; #P , 0.05 interaction; *compared with Veh of same condition; ¤compared with corresponding Sed of same treatment as assessed by two-way repeated-measures ANOVA with Sidak post hoc analysis. F: In vivo glucose uptake in HFD-fed mice after 6 days of compound A (Comp A) treatment, with 2-deoxy-D-glucose retro-orbital injection and submaximal insulin (0.5 units/kg). Data are mean 6 SEM for n =6–10 mice per group. ‡P , 0.05 main effect for UCN2 treatment; †P , 0.05 main effect for insulin; #P , 0.05 interaction; *compared with saline of the same treatment; ¤compared with Veh of the same condition as assessed by two-way ANOVA with Sidak post hoc analysis. Quad, quadriceps; Wheel, wheel running.

activation of CRHR2 with a modified UCN2 peptide re- to activating the CRHR2 peripherally with a PEGylated duced body weight in HFD-fed mice. These results are UCN2 compound, whereas genetic models/approaches primarily localized to peripheral tissues because the PEGy- represent a supraphysiological event that may not portray lated compound cannot cross the blood-brain barrier. the normal activity of the pathway. Our results also highlight Thus, strategies to activate CRHR2 in peripheral tissues potential discrepancies between activating CRHR2 with appear to have positive effects on energy homeostasis. In geneticmodelsfrombirthversustransientactivationof accordance with our results, Ucn2 adeno-associated virus CRHR2 with pharmacological treatments. gene transfer attenuates weight gain in HFD-fed mice Exercise and diet are considered a first-line treatment of (17). Our results represent a pharmacological approach insulin resistance and type 2 diabetes. For many patients, 1410 UCN2 and Skeletal Muscle Insulin Sensitivity Diabetes Volume 68, July 2019

Figure 3—CRHR2 agonist enhances skeletal muscle insulin signaling and glucose transport in HFD-fed mice. A, B, D: At the end of the treatment period, the EDL muscle was exposed ex vivo to a submaximal dose of insulin (0.36 nmol/L) for 1 h and Akt phosphorylation (pAkt) at Ser473 and Thr308, and total Akt was assessed from the lysates. C: Glucose transport into the EDL was also assessed. The dotted line indicates the mean of chow vehicle (Veh) mice. Data are mean 6 SEM for n =8–10 per group. ‡P , 0.05 main effect for UCN2 treatment; †P , 0.05 main effect for insulin; #P , 0.05 interaction; *compared with Veh of same condition; ¤compared with corresponding sedentary (Sed) of same treatment as assessed by two-way repeated-measures ANOVA with Sidak post hoc analysis. AU, arbitrary units; Comp A, compound A; Wheel, wheel running.

pharmacological intervention is required to manage this secretion, particularly in conditions of nutrient excess disease, yet effective insulin sensitizers are lacking from (28).Theoretically,theUCN2peptideusedheremay the current diabetes pharmacopeia. Here, we provide activate pancreatic b-cell CRHR2 and stimulate insulin evidence that UCN2 peptide treatment reduced fasting secretion; however, this remains to be determined. hyperglycemia and hyperinsulinemia in obese mice. The mechanistic basis of UCN2 treatment may involve Despite lower insulin levels during a GTT, compound enhanced insulin signaling. We have reported that insulin A treatment enhanced glucose tolerance during a GTT signaling and glucose transport are impaired in skeletal compared with vehicle-treated obese mice, indicating muscle from patients with type 2 diabetes (4,5). Thus, that UCN2 treatment improves insulin sensitivity. This is targeting components of the canonical insulin signaling consistent with an earlier study reporting that Ucn2 gene cascade or GLUT4 transport machinery in skeletal muscle transfer improves glycemia and insulin sensitivity in HFD- may improve glucose homeostasis (29,30). Indeed, we fed and db/db mice (17). Collectively, these results impli- found that UCN2 peptide treatment in HFD-fed mice cate peripheral action of UCN2 therapies for the treatment increased Akt phosphorylation and protein abundance of obesity and insulin resistance. Nevertheless, we cannot in skeletal muscle concomitant with enhanced insu- exclude the possibility that compound A acts on the pancreas. lin-stimulated glucose uptake. Consistent with this, tran- UCN3, but not UCN2, is expressed in the b-cells of the sient Ucn3 overexpression in skeletal muscle increases pancreas and acts in an autocrine manner on CRHR2 to protein abundance of IRS1, Akt, TBC1D4, and GSK3a/b regulate glucose-stimulated insulin production and (16). Insulin-stimulated TBC1D4 phosphorylation at Ser318 diabetes.diabetesjournals.org Borg and Associates 1411

Figure 4—Modified UCN2 increases GLUT4 translocation and glucose transport into skeletal muscle. A and B: L6-GLUT4-Myc myoblasts were stimulated with 100 nmol/L insulin, 100 nmol/L compound A (Comp A), or 100 nmol/L clenbuterol for 30 min, and GLUT4 translocation to the cell membrane was assessed (n = 6) with the accompanying representative images. *Compared with PBS assessed by one-way ANOVA. Soleus muscle was excised from chow-fed mice and incubated ex vivo with Comp A (63.3 nmol/L) in the absence or presence of a submaximal dose of insulin (0.18 nmol/L) for 1 h. C: Glucose transport into soleus. Conditions were as follows: basal (n = 10), insulin (n = 10), Comp A (n = 10), and Comp A plus insulin (n = 10). D–H: Representative Western blots for the assessment of phosphorylated Akt (p-Akt) at Ser473 and Thr308 and phosphorylated mTOR (p-mTOR) at Ser2448 and Ser2481. All samples were run on the same gel but in a different order. Data are mean 6 SEM. ‡P , 0.05 main effect for UCN2 treatment; †P , 0.05 main effect for insulin; #P , 0.05 interaction; *compared with vehicle (Veh) of same condition; ¤compared with corresponding sedentary of same treatment as assessed by two-way ANOVA with Sidak post hoc analysis. AU, arbitrary units. was increased in response to compound A treatment, while CRHR2 affects insulin signaling are unknown. Nonethe- phosphorylation at Thr642 was reduced compared with less, insulin-stimulated glucose uptake is increased in re- controls. However, the exact mechanisms by which sponse to either acute or chronic compound A treatment. 1412 UCN2 and Skeletal Muscle Insulin Sensitivity Diabetes Volume 68, July 2019

Moreover, we found that compound A acutely promoted the metabolic effect during the in vivo treatment that was GLUT4 translocation, which may account for the increased related to the reduced food intake is uncertain. Given the glucose clearance during the GTT as well as enhanced effect of compound A to reduce body weight, altered glucose uptake in isolated skeletal muscle. Improved glu- energy expenditure or thermogenesis could play a role. cose homeostasis by Ucn2 gene transfer in HFD-fed mice The hypothalamus is unlikely to be a direct target of was attributed to increased GLUT4 translocation (17). compound A because of the PEGylation, which results Along with increased skeletal muscle glucose uptake, gly- in poor blood-brain barrier drug penetration. Thus, any cogen content was unaltered (data not shown), suggesting potential neuroendocrine effect of compound A on energy that compound A improved glucose metabolism by in- homeostasis at the level of the hypothalamus is likely to creasing glucose oxidation. Thus, the enhanced glucose be secondary. However, without the inclusion of a PEG- uptake and metabolism in compound A–treated mice is not vehiclecontrol,wecannotfullyexcludethepossibilityof only due to a weight loss effect but also due to direct action a central component of compound A on the regulation of on skeletal muscle. energy homeostasis. We also do not believe that BAT is CRHR2 is differentially expressed in peripheral tissues, a major target of compound A because basal- or insulin- including cardiac and skeletal muscle (31), adipose tissue stimulated glucose uptake was unaltered. Nevertheless, (32), skin (9), and the gastrointestinal tract (33), where it we cannot exclude the possibility that non-insulin-mediated serves diverse functions. Activation of CRHR2 in the metabolic processes in BAT are affected. Skeletal muscle gastrointestinal tract is involved in gastric motility (34) appears to be a direct target of compound A. Compound A and intestinal inflammation (35), while activation in car- directly increases GLUT4 translocation in L6 cells and diac tissue is involved in blood pressure regulation (36). In increases insulin-stimulated glucose uptake and insulin the current study, subcutaneous administration of com- signaling in isolated soleus muscle from chow-fed mice. pound A targets CRHR2, which is present throughout the Additionally, electroporation of skeletal muscle with a UCN2 periphery and could therefore have numerous effects in plasmid increases glucose uptake. Thus, compound A has multiple organs controlling whole-body glucose and energy a direct and immediate effect on skeletal muscle metabolism homeostasis. Selective agonists for UCN2 and UCN3 re- independent of changes in adiposity. Our main findings duce gastric emptying (34,37). As such, we observed an related to the physiological effects of this approach to initial decrease in food intake and a corresponding re- control skeletal muscle insulin sensitivity and body weight duction in wheel running after the first day of treatment, is schematically highlighted (Fig. 5). which could be attributed to a decrease in gastric emptying CRHR2 activation in skeletal muscle enhances AMPK and the accompanying malaise. By the end of the treat- signaling, which increases glucose disposal (16,38) while ment period this effect was attenuated; however, without also activating AMPK in cardiac tissues (39). However, in the inclusion of a pair-fed control group, the proportion of the current study, AMPK and downstream signaling, such

PEG UCN2

CRHR2

Chronic Treatment Direct Modulaon

HFD HFD 14 days

↓ Body weight & food intake ↑ GLUT4 translocaon in L6 myoblasts ↓ Adiposity ↑ Ex vivo insulin-smulated glucose ↑ Glucose homeostasis uptake in soleus ↑ Skeletal muscle glucose uptake, ↑Insulin signaling in isolated soleus in vivo and ex vivo

Figure 5—Modified UCN2 regulates skeletal muscle insulin sensitivity. diabetes.diabetesjournals.org Borg and Associates 1413 as pACC (a surrogate marker for AMPK activation), was Institutet (Swedish Research Council grant number 2009-1068) supported this not altered (data not shown). In contrast, compound A research. M.L.B. is supported by the Swedish Society for Medical Research. treatment increased mTOR phosphorylation, implicating Duality of Interest. L.G., M.W., J.A.-F., R.M., A.R., S.B., T.C., E.O., E.M.N., a role in anabolic processes. A role for IGF-I signaling in and J.T.B. are employees of Eli Lilly. J.R.Z. received compound A as a gift from Eli Lilly. No other potential conflicts of interest relevant to this article were reported. UCN3-mediated hypertrophy of soleus, tibialis cranialis, Author Contributions. M.L.B., J.M., M.S., T.D.C.B., L.G., M.W., J.A.-F., and gastrocnemius muscle and glucose disposal has also R.M., A.R., S.B., T.C., E.O., E.M.N., A.V.C., H.K.K., and J.T.B. researched data. been proposed (15,16). However, plasma IGF-I levels after M.L.B., J.M., H.K.K., J.T.B., and J.R.Z. analyzed and interpreted the data. M.L.B., UCN2 treatment were unaltered (data not shown). Differ- H.K.K., J.T.B., and J.R.Z. designed the study. M.W., A.K., and J.T.B. contributed to ences between these studies may be accounted for by the the discussion and reviewed and edited the manuscript. M.L.B. and J.R.Z. wrote models studied (Ucn3 transgenic mice and overexpression the manuscript. All authors approved the manuscript. J.R.Z. is the guarantor of this in rats vs. subcutaneous injection in HFD mice) or the work and, as such, had full access to all the data in the study and takes specific ligand used to activate CRHR2 (UCN3 vs. UCN2), responsibility for the integrity of the data and the accuracy of the data analysis. resulting in different signaling/downstream effects. In References support of this, signaling through either CRHR or fi – 1. Hagberg JM, Coyle EF, Baldwin KM, et al. The historical context and scienti c G-protein coupled receptors confers distinct conforma- legacy of John O. Holloszy. J Appl Physiol (1985). 7 February 2019 [Epub ahead of tional changes, which elicit different coupling of the print]. DOI: 10.1152/japplphysiol.00669.2018 G-proteins and activation of signaling cascades (40–42). 2. Carpino PA, Goodwin B. Diabetes area participation analysis: a review of Specifically, UCN1 binding to CRHR1 or CRHR2 leads to companies and targets described in the 2008 - 2010 patent literature. 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