Modified UCN2 Peptide Acts as an Insulin Sensitizer inSkeletal Muscle of Obese MiceMelissa 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 andJuleen R. Zierath1,2

Diabetes 2019;68:1403–1414 | https://doi.org/10.2337/db18-1237

The neuropeptide urocortin 2 (UCN2) and its receptorcorticotropin-releasing hormone receptor 2 (CRHR2) arehighly expressed in skeletal muscle and play a role inregulating energy balance and glucose metabolism.We investigated a modified UCN2 peptide as a poten-tial therapeutic agent for the treatment of obesity andinsulin resistance, with a specific focus on skeletalmuscle. High-fat–fed mice (C57BL/6J) were injecteddaily with a PEGylated UCN2 peptide (compound A) at0.3 mg/kg subcutaneously for 14 days. Compound Areduced body weight, food intake, whole-body fat mass,and intramuscular triglycerides compared with vehicle-treated controls. Furthermore, whole-body glucose tol-erance was improved by compound A treatment, withincreased insulin-stimulated Akt phosphorylation atSer473 and Thr308 in skeletal muscle, concomitant withincreased glucose transport into extensor digitorum lon-gus and gastrocnemius muscle. Mechanistically, this islinked to a direct effect on skeletal muscle becauseex vivo exposure of soleus muscle from chow-fed leanmice to compound A increased glucose transport andinsulin signaling. Moreover, exposure of GLUT4-Myc–labeled L6 myoblasts to compound A increased GLUT4trafficking. Our results demonstrate that modified UCN2peptides may be efficacious in the treatment of type 2diabetes by acting as an insulin sensitizer in skeletalmuscle.

Exercise and diet are potent lifestyle interventions tocombat metabolic dysfunction by improving weight man-agement and glucose homeostasis. In particular, exercise

enhances skeletal muscle insulin sensitivity and mitochon-drial function (1). Nevertheless, such lifestyle interven-tions have poor adherence, requiring pharmacologicaladvances to alleviate obesity and prevent metabolic dis-ease. Consequently, efforts are under way to develop in-sulin sensitizers and weight-reducing pharmacologicalagents for the treatment of diabetes (2,3).

Skeletal muscle is an important tissue involved inmaintaining glucose homeostasis under insulin-stimulatedconditions and is a major site of insulin resistance in type2 diabetes (4,5). Although precise mechanisms of skeletalmuscle insulin resistance are not fully elucidated, impairedinsulin signaling and reduced glucose uptake are majoraspects (4,5). Insulin resistance is present at all pathogenicstages of type 2 diabetes progression. Consequently,efforts to maintain skeletal muscle insulin sensitivity toprevent/delay type 2 diabetes are warranted. In additionto lifestyle modifications, including diet and exercise, newtherapeutic routes to directly enhance skeletal muscleinsulin sensitivity, either as monotherapy or in combi-nation with other drugs, are of interest to treat type 2diabetes.

The corticotropin-releasing factor (CRF) urocortin(UCN) family of neuropeptides is a direct modulator ofthe hypothalamic-pituitary-adrenal axis both centrally andperipherally (6). Within this family are four peptides (CRFand UCN 1, 2, and 3) that are structurally related butencoded by separate genes (7). UCN peptides signalthrough two different G-protein–coupled receptors:corticotropin-releasing hormone receptors (CRHRs) 1

1Department of Physiology and Pharmacology, Section for Integrative Physiology,Karolinska Institutet, Stockholm, Sweden2Department of Molecular Medicine and Surgery, Section for Integrative Physi-ology, Karolinska Institutet, Stockholm, Sweden3Lilly Research Laboratories, Division of Eli Lilly and Company, Indianapolis, IN

Corresponding author: Juleen R. Zierath, juleen.zierath@ki.se

Received 21 November 2018 and accepted 8 April 2019

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db18-1237/-/DC1.

© 2019 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and the workis not altered. More information is available at http://www.diabetesjournals.org/content/license.

Diabetes Volume 68, July 2019 1403

METABOLISM

and 2 (8). These peptides and receptors are differentiallyexpressed in central and peripheral tissues (7,8). UCN1binds to both receptors, while UCN2 and UCN3 areselective for CRHR2. Skeletal muscle has high expressionlevels of both UCN2 and its receptor CRHR2 (9). Whileemerging evidence suggests that CRF peptides regulatecardiovascular and renal function and inflammatory pro-cesses (10), their role in metabolic diseases is unclear.

UCNs and CRHR2 play a role in glucose homeostasis.Ucn2 and Crhr2 knockout mice have enhanced glucosetolerance and increased insulin sensitivity and are pro-tected from high-fat diet (HFD)–induced obesity (11,12).HFD and/or elevated stress states upregulate skeletalmuscle CRHR2 (13), while CRHR2 activation inhibitsinsulin signaling (14). Thus, increased CRHR2 activityimpairs glucose homeostasis. In contrast, whole-bodyUcn3 transgenic mice are protected from HFD-inducedobesity (15), and transient overexpression of Ucn3 inskeletal muscle enhances glucose metabolism throughincreased insulin signaling (16). In addition, Ucn2 over-expression through systemic virus delivery improveswhole-body insulin sensitivity in HFD-fed rodents (17).Accordingly, activating CRHR2 during obesity can alsoenhance glucose homeostasis. While the UCN-CRHR axisappears to regulate skeletal muscle metabolism, the pre-dominant effects remain unclear.

Observations of aerobic training–like phenotypes intransgenic mice (18–20) has ignited interest in devel-oping pharmacological therapies to combat insulin re-sistance in patients with type 2 diabetes (21). Given therole of the UCN-CRHR axis in skeletal muscle metabo-lism, we hypothesized that UCN peptides act as insulinsensitizers in skeletal muscle. Thus, we investigated theeffects of a modified UCN2 peptide acting on the CHRH2in HFD-induced obese mice, with a specific focus onskeletal muscle.

RESEARCH DESIGN AND METHODS

Peptide SynthesisCompound A (a PEGylated peptide analog of humanUCN2) was synthesized using established solid-phase pep-tide synthesis protocols. After final cleavage of the peptidefrom the resin, the peptide was purified using reversed-phase chromatography and lyophilized to obtain peptidepowder as trifluoroacetate salt. The peptide was conju-gated to a 20-kDa functionalized polyethylene glycol (PEG)polymer through an acetamide-based linker. Formulatedaliquots of the peptide conjugate in PBS were storedat 220°C. Working solutions were freshly prepared fromthawed stock aliquots diluted with 0.5% pan-albumin/0.9%NaCl.

PharmacokineticsPharmacokinetics were determined in mice after a singlesubcutaneous administration of compound A. Plasma con-centrations of compound A were determined through

liquid chromatography with tandem mass spectrometry.Pharmacokinetic parameters were calculated by noncom-partmental analysis using Phoenix WinNonlin 6.3 software.

cAMP AssayHEK293 cells transfected with mouse CRHR1 or CRHR2bplasmid were plated in 96-well plates at 2,000 cells per welland allowed to attach overnight. Serial dilutions of hu-man UCN2 or compound A were placed onto the cellsfor 15 min. cAMP levels were measured using a cAMPcell-based assay kit (Cisbio).

L6-GLUT4-Myc Cell Surface DetectionL6 rat myoblasts expressing human GLUT4 with an exo-facial Myc-epitope tag were cultured in a 96-well plate andincubated in the absence or presence of 100 nmol/L in-sulin, 100 nmol/L compound A, or 100 nmol/L clenbuterolfor 30 min. Cell surface density of GLUT4-Myc was mea-sured as previously described (22). Fluorescence intensitywas obtained using a LI-COR Odyssey eXL (LI-COR Bio-sciences, Lincoln, NE).

AnimalsExperiments were approved by the Stockholm North an-imal ethics committee or the Eli Lilly institutional animalcare and use committee. Male mice (C57BL/6J) werepurchased from Charles River Laboratories (Sulzfeld, Ger-many) or Envigo (Somerset, NJ) at 5 weeks of age. Micewere maintained under a 12-h light/dark cycle and had freeaccess to water and standard rodent chow (4% kcal fromfat, R34; Lantmännen, Kimstad, Sweden). At 6 weeks ofage, mice were placed on either a standard rodent chow oran HFD (60% kcal from fat, TD.06414; Harlan Laborato-ries) ad libitum for 20 weeks and were single housed after19 weeks. After 20 weeks on an HFD, mice received dailysubcutaneous injections of vehicle before the onset of thedark period (0.5% pan-albumin/0.9% NaCl) or compound A(0.3 mg/kg body weight) for 14 days. Injections wereperformed in the intrascapular region or hind leg onalternating days to minimize discomfort.

Free Wheel RunningHFD-fed mice were randomized into sedentary or wheelrunning groups. Wheel running mice were acclimatized tothe running wheels for 7 days, and all groups were weightand running matched before injections. Body weight andfood intake were recorded daily. Activity of the mice on therunning wheels (35-cm diameter) was monitored by a mag-netic switch affixed to each wheel, which recorded thenumber of revolutions. Data were captured by an automatedcomputer monitoring system (VitalView application soft-ware; Mini-Mitter Company). Physical activity was recordedcontinuously as wheel revolutions per 5-min interval.

Ex Vivo Glucose UptakeExtensor digitorum longus (EDL) muscles were dissectedfrom 4-h–fasted mice anesthetized with an intraperitoneal

1404 UCN2 and Skeletal Muscle Insulin Sensitivity Diabetes Volume 68, July 2019

injection of 16mL/g body weight 2.5% 2,2,2-tribromoethanoland tertiary amyl alcohol. Muscles were incubated withKrebs-Henseleit buffer under continuous gassing (95%O2/5% CO2) at 30°C in the absence (basal) or presence of0.36 nmol/L insulin (Actrapid; Novo Nordisk), and2-deoxy-D-glucose uptake was determined as previ-ously described (23). Results are expressed as mmol/Lglucose 3 mg protein21 3 20 min21.

Soleus muscles from 8-week-old, chow-fed mice wereincubated in the absence or presence of compound A(63.3 nmol/L) with or without a submaximal insulin dose(0.18 nmol/L) to assess insulin sensitivity. Glucose uptakewas determined as described above.

In Vivo Glucose UptakeMice fed an HFD for 20 weeks received daily subcutaneousinjections of compound A (0.3 mg/kg) or vehicle for 6 days.Fasted mice (4 h) were anesthetized with isoflurane. Micereceived 10 mCi [3H]2-deoxy-D-glucose (PerkinElmer) 60.5 units/kg insulin (Humilin R; Eli Lilly) by retro-orbitalinjection. Blood samples were taken at 2, 5, 10, 15, 20, and30 min after injection, treated with Ba(OH)2, and pre-cipitated with ZnSO4 for determination of blood-specificactivity. After centrifugation, the supernatant was col-lected, and radioactivity was determined using liquidscintillation counting (Beckman LSC). Animals were eu-thanized, and tissues were frozen. Tissue homogenateswere mixed with either water to determine total 2-deoxy-D-glucose or Ba(OH)2/ZnSO4 to determine unphosphory-lated 2-deoxy-D-glucose as previously described (24).

Glucose Tolerance and Body CompositionGlucose tolerance and body composition were determinedon day 11 of the treatment. Glucose (2 g/kg body weight)was administered by intraperitoneal injection in 4-h–fasted mice. Blood was sampled through the tail vein toassess glucose (OneTouch Ultra 2 glucose meter; LifeScan)and insulin (Insulin ELISA Kit; Crystal Chem). Total leanand fat mass was assessed in conscious mice using theEchoMRI-100 system (Echo Medical Systems).

Electroporation StudyChow-fed male mice (7–9 weeks of age) were anesthetizedwith isoflurane and a solution of 100 mL of hyaluronidase(Sigma H-3506) (2 mg/mL in Tyrode’s buffer) was injectedinto the triceps surae and tibialis anterior (TA) (twoseparate injections) in each leg 1 h before the DNA in-jection. Mice were then injected intramuscularly in thetibialis and triceps surae with 100 mg human UCN2plasmid construct (catalog number RC201333, RefSeqNM_033199.3; Origene) (two separate injections; 1 mg/mLin Tris-EDTA [TE] buffer) and an equal amount of TEbuffer in the contralateral leg as a control. Thereafter, theleg was subjected to electroporation (mode LV, 99 ms/500 V,voltage 150 V, four 20-ms pulses one per second,150 V/cm) using a BTX 830M electroporation unit(BTX, Holliston, MA) fitted with gene caliper electrodes

(BTX). Four days after electroporation, mice were sub-jected to in vivo glucose uptake.

Biochemical AnalysisGlycogen and triacylglyceride (TAG) content in liver andTA muscle were measured in 4-h–fasted mice using a Gly-cogen Assay Kit (ab65620; Abcam) or Triglyceride Quan-tification Assay Kit (ab65336; Abcam) according to themanufacturer’s protocol. Plasma free fatty acid (ab65341;Abcam) and plasma leptin (MOB00; R&D Systems) wereanalyzed using assay kits according to the manufacturers’protocol.

Western Blot AnalysisWestern blot analysis was performed as previously de-scribed (25). Primary antibodies used are listed in Supple-mentary Table 1. Bands were quantified using QuantityOne 1-D analysis software (Bio-Rad) and normalized tototal protein staining with Ponceau S (Sigma-Aldrich).

StatisticsSignificant differences were determined by one-way, two-way, or two-way repeated-measures ANOVA with Sidakmultiple comparison post hoc test as indicated in the figurelegends. Chow-fed mice were excluded from statisticalanalysis because they served as a control for the HFD.Comparisons were considered significant at P , 0.05.Analyses were performed using GraphPad 7 statisticalsoftware (GraphPad Software).

RESULTS

Characteristics of the Modified UCN2 PeptideThe modified human UCN2 peptide (compound A) is basedon the previously reported compound 8 (26). In contrast tocompound 8, compound A includes a cysteine residue atposition 29, where a PEG 20,000 is attached through anacetamide-based linker. The potency of compound A wasassessed by cAMP production in HEK293 cells transfectedwith mouse CRHR1 or CRHR2b plasmid (the predominantskeletal muscle isoform) (Table 1). CRHR2b-transfectedcells treated with serial dilutions of compound A for15 min had a half-maximal effective concentration valueof 0.31 nmol/L compared with 0.08 nmol/L for humanUCN2, while there was no cAMP production in cells trans-fected with CRHR1. Time to maximum plasma concentra-tion after mice were treated with a single subcutaneousinjection of compound A was;4 h, while the clearance was

Table 1—EC50 of response elicited by peptides

Peptide CRHR1 EC50 CRHR2b EC50

Human UCN2 .10,000 (no activity) 0.08 6 0.03

Compound A .10,000 (no activity) 0.31 6 0.01

Data are mean nmol/L6 SD of compound added. The biologicalactivity of human UCN2 or compound A was assessed bydetermining cAMP production to serial dilutions. EC50,half-maximal effective concentration.

diabetes.diabetesjournals.org Borg and Associates 1405

estimated at 5.94 mL/h/kg. Compound A has a half-lifeof 22.3 h compared with 15 min for the native UCN2peptide (27).

Chronic Activation of CRHR2 With Modified UCN2Reverses HFD-Induced ObesityTo investigate the metabolic effects of modified UCN2peptide, male mice were fed an HFD for 20 weeks beforedaily subcutaneous injections of vehicle or compound A(0.3 mg/kg) for 14 days. Body weight was reduced incompound A–treated mice after day 1, with a cumulativeweight loss of 7.5 g over the 14-day treatment period (Fig.1A and B and Table 2). Weight loss in compound A–treatedmice was accompanied by an 84% initial reduction in foodintake compared with controls, which was followed bya 28% decrease at the end of the treatment period (Fig. 1Cand Table 2). Thus, we noted a large decrease in food intakein the first several days and a smaller, but still significantdecrease later in the treatment. The reduction in foodintake after the first injection with compound A is likelydue to an initial reduction in gastric emptying wherebya single compound A injection reduced gastric emptying by52% compared with vehicle (Supplementary Fig. 1). Feedefficiency (the ratio of body weight change to food intake)was initially reduced 34% with compound A treatment,which was followed by a significant reduction at the end ofthe treatment period (Fig. 1D). Weight loss in compoundA–treated mice was attributed to a decrease in fat masswithout alteration in lean mass (Fig. 1E and Table 2).Compound A treatment reduced TAG content in TAmusclecompared with vehicle-treated mice (Fig. 1F). Compound Atreatment reduced liver weight without altering hepaticTAG or glycogen content (Table 1).

To investigate whether compound A treatment hasa synergistic effect with physical activity, mice were givenfree access to running wheels over the 14-day treatmentperiod. In vehicle-treated mice, wheel running reducedbody fat mass (Fig. 1E) without altering lean mass (Table2), and this was associated with reduced final body weightcompared with sedentary mice (Table 2). Wheel runningreduced the absolute and percent weight loss (Fig. 1A andB) and final body weight in compound A–treated micecompared with vehicle-treated wheel running mice (Table2), despite less distance ran (Supplementary Fig. 2). Theweight loss was attributed to decreased fat mass (Fig. 1E)without altering lean mass, liver weight (Table 2), orTA TAGs (Fig. 1F) compared with vehicle-treated wheelrunning mice. There were no synergistic effects with wheelrunning in the phenotypic improvements seen withcompound A treatment alone. This highlights the potentnature of compound A on weight loss and fat massreduction.

Chronic Activation of CRHR2 With Modified UCN2Improves In Vivo Glucose HomeostasisNext, we investigated the whole-body glucose homeostasisof compound A–treated mice. Compound A–treated mice

had reduced fasting blood glucose (Fig. 2A) and fastingplasma insulin (Fig. 2B) compared with HFD-fed vehicle-treated mice. Despite reduced plasma insulin (Fig. 2C),compound A–treated mice cleared the same amount ofblood glucose as vehicle-treated control mice during a glu-cose tolerance test (GTT) (Fig. 2D and E). Moreover, in vivoinsulin-stimulated glucose uptake into red and whitequadriceps and EDL from compound A–treated micewas enhanced compared with insulin-stimulated vehicle-treated control muscles, while there was no alteration ininsulin-stimulated glucose uptake in brown adipose tissue(BAT) (Fig. 2F).

Although wheel running in vehicle-treated mice did notaffect fasting blood glucose levels compared with seden-tary vehicle-treated mice (Fig. 2A), it reduced fastingplasma insulin levels (Fig. 2B). Wheel running vehicle-treated mice cleared the same amount of glucose duringa GTT as sedentary vehicle-treated mice (Fig. 2D and E),even with reduced plasma insulin (Fig. 2C). These datasuggest that wheel running improves insulin sensitivity inHFD-fed mice.

Wheel running in compound A–treated mice reducedfasting blood glucose (Fig. 2A) and fasting plasma insulin(Fig. 2B) compared with vehicle-treated wheel runningmice. Blood glucose clearance was increased in compoundA–treated wheel running mice compared with wheel run-ning controls (Fig. 2D and E), although similar plasmainsulin levels were observed during the GTT (Fig. 2C). Wefound no differences between compound A–treated sed-entary and compound A–treated wheel runningmice in theaforementioned parameters of in vivo glucose homeosta-sis. Thus, while wheel running in vehicle-treated miceimproved glucose homeostasis, compound A treatmentalone was more potent, supporting the notion thatUCN2 peptide treatment in HFD-fed mice improves skel-etal muscle and whole-body insulin sensitivity with in-creased glucose uptake.

Chronic Activation of CRHR2 With Modified UCN2Improves Skeletal Muscle Insulin SensitivityTo further investigate potential mechanisms of action forthe positive effect of compound A on whole-body glucosehomeostasis, we assessed insulin sensitivity of EDL muscleex vivo after compound treatment. Insulin-stimulated Aktphosphorylation at Ser473 and Thr308 was increased in EDLfrom compound A–treated mice compared with vehicle-treated mice (Fig. 3A and B). TBC1D4 phosphorylation atSer318 was increased above basal levels in compound A–treated EDL muscle (Supplementary Fig. 3A), whereasinsulin-stimulated phosphorylation at Thr642 was de-creased compared with controls (Supplementary Fig.3B). A trend for increased insulin-stimulated GSK3a phos-phorylation at Ser21 was observed in EDL muscle fromcompound A–treated mice compared with vehicle-treatedEDL (P = 0.087) (Supplementary Fig. 3C). Total Akt proteinwas increased in EDL muscle from compound A–treatedmice (Fig. 3D), whereas protein abundance of downstream

1406 UCN2 and Skeletal Muscle Insulin Sensitivity Diabetes Volume 68, July 2019

Figure 1—CRHR2 agonist reduces body weight (BW), food intake, and fat mass in HFD-fedmice. The following groups were studied: vehiclesedentary (Veh) (n = 9), vehicle wheel running (VehWheel) (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 andB: BW expressed as absolute and percent change from the first day of subcutaneous injections in sedentary mice and mice exposedto voluntary wheel running for the duration of the treatment. C and D: Food intake and feed efficiency (calculated by the ratio between BWloss 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; *CompA vs. Veh; ^Comp AWheel vs. VehWheel. E: MRI was performed on day 11 of treatment tomeasure total fat mass. F:TAG content of the TA. Dotted line indicates the mean of Chow Veh mice. Data are mean6 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 sametreatment as assessed by two-way ANOVA with Sidak post hoc analysis. BW and food intake assessed by two-way repeated-measuresANOVA with Sidak post hoc analysis. Sed, sedentary.

diabetes.diabetesjournals.org Borg and Associates 1407

targets, such as GLUT 4, GSK3a, GSK3b, or glycogensynthase, were unaltered (Supplementary Fig. 3D–F).Insulin-stimulated glucose transport into the EDL musclewas increased in compound A–treated mice compared withvehicle-treated mice (Fig. 3C).

Acute Activation of CRHR2 With Modified UCN2Directly Enhances GLUT4 Translocation in L6Myoblasts and Insulin Sensitivity in SoleusSkeletal MuscleTo assess the potential direct effect of UCN2 on skeletalmuscle metabolism, without the confounding factors ofreduced adiposity and food intake with in vivo compoundA treatment, TA and triceps surae muscles from lean chow-fed mice were electroporated with vectors containinghuman UCN2 or TE buffer in the contralateral leg. Ex-pression of human UCN2 was detected in TA and soleusmuscle after transfection with human UCN2 vectors (Sup-plementary Fig. 4A). UCN2 overexpression in mouse mus-cle was associated with a modest reduction in endogenousUcn2 expression (Supplementary Fig. 4A). UCN2 overex-pression increased glucose transport into soleus musclecompared with the control contralateral leg (Supplemen-tary Fig. 4B), confirming a positive regulation of skeletalmuscle glucose transport by UCN2. In L6-GLUT4-Mycmyoblasts, compound A (100 nmol/L) enhanced GLUT4translocation to the membrane to levels comparable toinsulin stimulation (100 nmol/L) (Fig. 4A and B). Com-pound A stimulation also increased glucose uptake ex vivoin skeletal muscle. Isolated soleus muscle from lean chow-fed mice was incubated with compound A (63.3 nmol/L),with or without submaximal insulin (0.18 nmol/L) for 1 h.Compound A increased insulin-stimulated glucose uptakeinto soleus muscle compared with insulin-stimulated ve-hicle treatment (Fig. 4C). Akt phosphorylation at bothSer473 and Thr308 was increased in response to insulin andcompound A stimulation compared with insulin stimu-lation alone (Fig. 4C–E). Mammalian target of rapamycin

(mTOR) phosphorylation at Ser2448 and Ser2481 was in-creased in response to compound A in an insulin-independent manner (Fig. 4D, G, and H).

DISCUSSION

In the context of changing demographic patterns and theaging population, current pharmacological treatments tocombat the majority of lifestyle-related conditions areinadequate. In particular, pharmacological treatmentsfor type 2 diabetes that specifically target skeletal muscleto increase insulin sensitivity and preserve skeletal musclefunction are lacking. In this regard, CRHR2 agonists mayimprove skeletal muscle substrate metabolism and miti-gate aging-associated disorders. Here, we determinedthe effects of a modified UCN2 peptide in HFD, obesemice. We show that compound A treatment of HFD-fedmice results in an initial reduction in food intake and rapidweight loss, which was accompanied by improved whole-body glucose tolerance and insulin-stimulated glucoseuptake into skeletal muscle. Mechanistically, this couldbe due to an effect on skeletal muscle because ex vivostimulation of soleus muscle from lean chow-fed mice withcompound A increased glucose uptake and insulin signal-ing. Thus, UCN2 peptides may be efficacious in the treat-ment of type 2 diabetes by acting as insulin sensitizers.

Genetic manipulation of CRF family members altersbody weight in mouse models. While body weight in Ucn2knockout mice is unaltered after 16 weeks on an HFD, fatmass is reduced, and lean mass is increased (11). Con-versely, overexpression ofUcn3, which also signals throughCRHR2, increases body weight, with increased lean massin chow-fed transgenic mice, whereas the HFD-fed trans-genic mice are obesity resistant (15). These genetic modelsare at the whole-body level, and therefore, the contributionof a centrally mediated effect on metabolism cannot beexcluded. Here, we provide evidence that pharmacological

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 mean6 SEM for n = 8–10 mice per group. Phenotypic characteristics of vehicle- or compound A–treated mice over the 14-daytreatment period. Mice were housed individually in cages without (sedentary) or with wheel running. ‡P , 0.05. Main effect for UCN2treatment. †P , 0.05. Main effect for wheel running. *Compared with vehicle of same condition. ¤Compared with correspondingsedentary of same treatment as assessed by two-way ANOVA with Sidak post hoc analysis.

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activation of CRHR2 with a modified UCN2 peptide re-duced body weight in HFD-fed mice. These results areprimarily localized to peripheral tissues because the PEGy-lated compound cannot cross the blood-brain barrier.Thus, strategies to activate CRHR2 in peripheral tissuesappear to have positive effects on energy homeostasis. Inaccordance with our results, Ucn2 adeno-associated virusgene transfer attenuates weight gain in HFD-fed mice(17). Our results represent a pharmacological approach

to activating the CRHR2 peripherally with a PEGylatedUCN2 compound, whereas genetic models/approachesrepresent a supraphysiological event that may not portraythe normal activity of the pathway. Our results also highlightpotential discrepancies between activating CRHR2 withgenetic models from birth versus transient activation ofCRHR2 with pharmacological treatments.

Exercise and diet are considered a first-line treatment ofinsulin resistance and type 2 diabetes. For many patients,

Figure 2—CRHR2 agonist improves glucose homeostasis in HFD-fed mice. A and B: Fasting blood glucose and fasting plasma insulin. Anintraperitoneal 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 correspondingsedentary (Sed) of same treatment as assessed by two-wayANOVAwith Sidak post hoc analysis. E: Blood glucose during theGTT. ‡P, 0.05main effect for UCN2 treatment; uP , 0.05 main effect for time; #P , 0.05 interaction; *compared with Veh of same condition; ¤comparedwith corresponding Sed of same treatment as assessed by two-way repeated-measures ANOVA with Sidak post hoc analysis. F: In vivoglucose uptake in HFD-fed mice after 6 days of compound A (Comp A) treatment, with 2-deoxy-D-glucose retro-orbital injection andsubmaximal insulin (0.5 units/kg). Data are mean6 SEM for n = 6–10 mice per group. ‡P, 0.05 main effect for UCN2 treatment; †P, 0.05main effect for insulin; #P , 0.05 interaction; *compared with saline of the same treatment; ¤compared with Veh of the same condition asassessed by two-way ANOVA with Sidak post hoc analysis. Quad, quadriceps; Wheel, wheel running.

diabetes.diabetesjournals.org Borg and Associates 1409

pharmacological intervention is required to manage thisdisease, yet effective insulin sensitizers are lacking fromthe current diabetes pharmacopeia. Here, we provideevidence that UCN2 peptide treatment reduced fastinghyperglycemia and hyperinsulinemia in obese mice.Despite lower insulin levels during a GTT, compoundA treatment enhanced glucose tolerance during a GTTcompared with vehicle-treated obese mice, indicatingthat UCN2 treatment improves insulin sensitivity. This isconsistent with an earlier study reporting that Ucn2 genetransfer improves glycemia and insulin sensitivity in HFD-fed and db/db mice (17). Collectively, these results impli-cate peripheral action of UCN2 therapies for the treatmentof obesity and insulin resistance. Nevertheless, we cannotexclude the possibility that compound A acts on the pancreas.UCN3, but not UCN2, is expressed in the b-cells of thepancreas and acts in an autocrine manner on CRHR2 toregulate glucose-stimulated insulin production and

secretion, particularly in conditions of nutrient excess(28). Theoretically, the UCN2 peptide used here mayactivate pancreatic b-cell CRHR2 and stimulate insulinsecretion; however, this remains to be determined.

The mechanistic basis of UCN2 treatment may involveenhanced insulin signaling. We have reported that insulinsignaling and glucose transport are impaired in skeletalmuscle from patients with type 2 diabetes (4,5). Thus,targeting components of the canonical insulin signalingcascade or GLUT4 transport machinery in skeletal musclemay improve glucose homeostasis (29,30). Indeed, wefound that UCN2 peptide treatment in HFD-fed miceincreased Akt phosphorylation and protein abundancein skeletal muscle concomitant with enhanced insu-lin-stimulated glucose uptake. Consistent with this, tran-sient Ucn3 overexpression in skeletal muscle increasesprotein abundance of IRS1, Akt, TBC1D4, and GSK3a/b(16). Insulin-stimulated TBC1D4 phosphorylation at Ser318

Figure 3—CRHR2 agonist enhances skeletal muscle insulin signaling and glucose transport in HFD-fed mice. A, B, D: At the end of thetreatment period, the EDLmuscle was exposed ex vivo to a submaximal dose of insulin (0.36 nmol/L) for 1 h and Akt phosphorylation (pAkt) atSer473 and Thr308, and total Akt was assessed from the lysates. C: Glucose transport into the EDL was also assessed. The dotted lineindicates themean of chow vehicle (Veh) mice. Data aremean6SEM for n = 8–10 per group. ‡P, 0.05main 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) ofsame treatment as assessed by two-way repeated-measures ANOVA with Sidak post hoc analysis. AU, arbitrary units; Comp A, compoundA; Wheel, wheel running.

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was increased in response to compound A treatment, whilephosphorylation at Thr642 was reduced compared withcontrols. However, the exact mechanisms by which

CRHR2 affects insulin signaling are unknown. Nonethe-less, insulin-stimulated glucose uptake is increased in re-sponse to either acute or chronic compound A treatment.

Figure 4—Modified UCN2 increases GLUT4 translocation and glucose transport into skeletal muscle. A and B: L6-GLUT4-Myc myoblastswere stimulatedwith 100 nmol/L insulin, 100 nmol/L compound A (CompA), or 100 nmol/L clenbuterol for 30min, andGLUT4 translocation tothe 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 ofa 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) atSer473 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 withvehicle (Veh) of same condition; ¤compared with corresponding sedentary of same treatment as assessed by two-way ANOVA with Sidakpost hoc analysis. AU, arbitrary units.

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Moreover, we found that compound A acutely promotedGLUT4 translocation, which may account for the increasedglucose clearance during the GTT as well as enhancedglucose uptake in isolated skeletal muscle. Improved glu-cose homeostasis by Ucn2 gene transfer in HFD-fed micewas attributed to increased GLUT4 translocation (17).Along with increased skeletal muscle glucose uptake, gly-cogen content was unaltered (data not shown), suggestingthat compound A improved glucose metabolism by in-creasing glucose oxidation. Thus, the enhanced glucoseuptake andmetabolism in compound A–treated mice is notonly due to a weight loss effect but also due to direct actionon skeletal muscle.

CRHR2 is differentially expressed in peripheral tissues,including cardiac and skeletal muscle (31), adipose tissue(32), skin (9), and the gastrointestinal tract (33), where itserves diverse functions. Activation of CRHR2 in thegastrointestinal tract is involved in gastric motility (34)and intestinal inflammation (35), while activation in car-diac tissue is involved in blood pressure regulation (36). Inthe current study, subcutaneous administration of com-pound A targets CRHR2, which is present throughout theperiphery and could therefore have numerous effects inmultiple organs controlling whole-body glucose and energyhomeostasis. Selective agonists for UCN2 and UCN3 re-duce gastric emptying (34,37). As such, we observed aninitial decrease in food intake and a corresponding re-duction in wheel running after the first day of treatment,which could be attributed to a decrease in gastric emptyingand the accompanying malaise. By the end of the treat-ment period this effect was attenuated; however, withoutthe inclusion of a pair-fed control group, the proportion of

the metabolic effect during the in vivo treatment that wasrelated to the reduced food intake is uncertain. Given theeffect of compound A to reduce body weight, alteredenergy expenditure or thermogenesis could play a role.The hypothalamus is unlikely to be a direct target ofcompound A because of the PEGylation, which resultsin poor blood-brain barrier drug penetration. Thus, anypotential neuroendocrine effect of compound A on energyhomeostasis at the level of the hypothalamus is likely tobe secondary. However, without the inclusion of a PEG-vehicle control, we cannot fully exclude the possibility ofa central component of compound A on the regulation ofenergy homeostasis. We also do not believe that BAT isa major target of compound A because basal- or insulin-stimulated glucose uptake was unaltered. Nevertheless,we cannot exclude the possibility that non-insulin-mediatedmetabolic processes in BAT are affected. Skeletal muscleappears to be a direct target of compound A. Compound Adirectly increases GLUT4 translocation in L6 cells andincreases insulin-stimulated glucose uptake and insulinsignaling in isolated soleus muscle from chow-fed mice.Additionally, electroporation of skeletal muscle with aUCN2plasmid increases glucose uptake. Thus, compound A hasa direct and immediate effect on skeletal musclemetabolismindependent of changes in adiposity. Our main findingsrelated to the physiological effects of this approach tocontrol skeletal muscle insulin sensitivity and body weightis schematically highlighted (Fig. 5).

CRHR2 activation in skeletal muscle enhances AMPKsignaling, which increases glucose disposal (16,38) whilealso activating AMPK in cardiac tissues (39). However, inthe current study, AMPK and downstream signaling, such

UCN2

Chronic Treatment Direct Modula�on

↓ Body weight & food intake↓ Adiposity↑ Glucose homeostasis ↑ Skeletal muscle glucose uptake,

in vivo and ex vivo

↑ GLUT4 transloca�on in L6 myoblasts ↑ Ex vivo insulin-s�mulated glucose uptake in soleus ↑Insulin signaling in isolated soleus

14 days

CRHR2

HFD

PEG

HFD

Figure 5—Modified UCN2 regulates skeletal muscle insulin sensitivity.

1412 UCN2 and Skeletal Muscle Insulin Sensitivity Diabetes Volume 68, July 2019

as pACC (a surrogate marker for AMPK activation), wasnot altered (data not shown). In contrast, compound Atreatment increased mTOR phosphorylation, implicatinga role in anabolic processes. A role for IGF-I signaling inUCN3-mediated hypertrophy of soleus, tibialis cranialis,and gastrocnemius muscle and glucose disposal has alsobeen proposed (15,16). However, plasma IGF-I levels afterUCN2 treatment were unaltered (data not shown). Differ-ences between these studies may be accounted for by themodels studied (Ucn3 transgenic mice and overexpressionin rats vs. subcutaneous injection in HFD mice) or thespecific ligand used to activate CRHR2 (UCN3 vs. UCN2),resulting in different signaling/downstream effects. Insupport of this, signaling through either CRHR orG-protein–coupled receptors confers distinct conforma-tional changes, which elicit different coupling of theG-proteins and activation of signaling cascades (40–42).Specifically, UCN1 binding to CRHR1 or CRHR2 leads toCREB and mitogen-activated protein kinase phosphoryla-tion, whereas CRF binding does not (43,44). Furthermore,Ucn2 gene transfer increases glucose disposal in mice,while Ucn3 gene transfer has no effect (45). Thus, theuse of specific ligands may fine-tune specific effects onmetabolic or gene regulatory pathways to influence glucoseor energy homeostasis.

Exercise training increases insulin sensitivity and glu-cose uptake in skeletal muscle of obese patients andprevents type 2 diabetes progression (46,47). We deter-mined whether UCN2 treatment and voluntary wheelrunning have a synergistic effect on skeletal muscle insulinsensitivity. In vehicle-treated obese mice, wheel runningreduced hyperinsulinemia and increased insulin sensitivityduring a GTT, while addition of compound A treatmentproduced negligible effects over the treatment alone. Thus,increased physical activity does not further enhance theinsulin sensitizing effects of compound A possibly becauseof the potent nature of compound A treatment alone.

G-protein–coupled receptors are the target of manymodern pharmaceutical drugs. There are currently nopharmacological agents that target skeletal muscle forthe treatment of type 2 diabetes. An agent that notonly increases skeletal muscle insulin sensitivity but alsoreduces body weight would be highly desired to treat thegrowing metabolically perturbed population. Indeed, acuteUCN2 peptide infusion is currently being tested clinicallyas an adjunct treatment in patients with heart failure(48–50), although a treatment for type 2 diabetes requiresa more long-term regimen. In conclusion, our results filla therapeutic void by providing new evidence for a treat-ment for type 2 diabetes that acts on skeletal muscle toenhance insulin sensitivity and glucose transport.

Funding. Vetenskapsrådet (Swedish Research Council) (2011-3550, 2015-00165); Swedish Diabetes Foundation (DIA2015-032); Stiftelsen för StrategiskForskning (Swedish Foundation for Strategic Research) (SRL10-0027); and NovoNordisk Foundation, Strategic Research Programme in Diabetes, at Karolinska

Institutet (Swedish Research Council grant number 2009-1068) supported thisresearch. M.L.B. is supported by the Swedish Society for Medical Research.Duality of Interest. L.G., M.W., J.A.-F., R.M., A.R., S.B., T.C., E.O., E.M.N.,and J.T.B. are employees of Eli Lilly. J.R.Z. received compound A as a gift from EliLilly. No other potential conflicts of interest relevant to this article were reported.Author Contributions. M.L.B., J.M., M.S., T.D.C.B., L.G., M.W., J.A.-F.,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.M.L.B., J.M., H.K.K., J.T.B., and J.R.Z. analyzed and interpreted the data. M.L.B.,H.K.K., J.T.B., and J.R.Z. designed the study. M.W., A.K., and J.T.B. contributed tothe discussion and reviewed and edited the manuscript. M.L.B. and J.R.Z. wrotethe manuscript. All authors approved the manuscript. J.R.Z. is the guarantor of thiswork and, as such, had full access to all the data in the study and takesresponsibility for the integrity of the data and the accuracy of the data analysis.

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