Mouse body composition analysis

Body composition of myonectin-KO mice and WT littermates was determined using a quantitative NMR instrument (Echo-MRI-100; Echo Medical Systems, Houston, TX, USA) at the Johns Hopkins University School of Medicine mouse phenotyping core facility. Echo-MRI analyses measured total fat mass, lean mass, and water content.

Indirect calorimetry

Indirect calorimetry was performed as previously described by Petersen et al. (54). In brief, WT and myonectin-KO mice were used for simultaneous assessments of daily body weight change, food intake (corrected for spillage), physical activity, and whole-body metabolic profile in an open-flow indirect calorimeter (Comprehensive Lab Animal Monitoring System; Columbus Instruments, Columbus, OH, USA). Data were collected for 3–4 consecutive d to confirm that mice were acclimated to calorimetry chambers (indicated by stable body weights, food intakes, and diurnal metabolic patterns), and data from the following days were analyzed. Rates of Vo₂ and Vco₂ in each chamber were measured throughout the studies. Respiratory exchange ratio (RER; Vco₂/Vo₂) was calculated by Comprehensive Lab Animal Monitoring System software (v.4.02) to estimate relative oxidation of carbohydrates (RER = 1.0) vs. fats (RER = 0.7), not accounting for protein oxidation. Energy expenditure (EE) was calculated as EE = Vo₂ × [3.815 + (1.232 × RER)]. Vo₂, Vco₂, and EE data were normalized to lean mass. Physical activity was measured by infrared beam breaks in the metabolic chamber. Mean metabolic values were calculated per subject and averaged across subjects for statistical analysis.

Mouse serum and blood chemistry analysis

Blood samples were collected by tail bleeds using capillary blood collection tubes (Microvette CB 300; Sarstedt, Nümbrecht, Germany). Blood samples were allowed to clot on ice and then centrifuged for 10 min at 10,000 g to collect serum. Serum triglyceride (TG) and cholesterol levels were measured using an Infinity kit (Thermo Fisher Scientific, Waltham, MA, USA). Nonesterified free fatty acids (NEFAs) were measured using a NEFA-HR (2) kit (Fujifilm, Tokyo, Japan). β-Hydroxybutyrate was measured using a LiquiColor assay (EKF Diagnostics, Cardiff, United Kingdom). Assays were performed according to the manufacturer’s protocol.

ELISA

Insulin (MilliporeSigma, Burlington, MA, USA), angiopoietin-like protein (ANGPTL) 3 (Thermo Fisher Scientific), and ANGPTL4 (Aviva Systems Biology, San Diego, CA, USA) levels were measured in serum according to the manufacturer’s protocol.

Glucose and insulin tolerance tests

For glucose tolerance tests, mice were unfed overnight (~15 h) and subsequently intraperitoneally injected or orally gavaged with a glucose solution in saline at a dose of 1 g glucose/kg body weight. Blood was collected from the tail vein into capillary blood collection tubes before injection and 15 min postinjection for assessment of insulin levels. Serum was isolated from blood and stored at −80°C until analysis. Tail vein blood glucose levels at the indicated time points were monitored with a glucometer (Nova Max Plus, Nova Biomedical, Waltham, MA, USA). For insulin tolerance tests (ITTs), mice were intraperitoneally injected with recombinant human insulin (Thermo Fisher Scientific) diluted in saline after a 2-h unfed period at a dose of 1–1.5 U insulin/kg body weight. Food was removed between 9:00 am and 10:00 am. Tail vein blood glucose was monitored with a glucometer at the indicated time points.

Lipid tolerance test

After an overnight unfed period (~15 h), mice were orally gavaged with 20% emulsified intralipid (soybean oil; MilliporeSigma) at a dose of 10 μl/g body weight. Blood was collected from the tail vein into capillary blood collection tubes before gavage and 1, 2, 3, and 4 h postgavage. Serum was isolated from blood samples and assayed for TG and NEFAs using commercially available kits according to the manufacturer’s protocol.

Hepatic VLDL–TG secretion

Mice were unfed for 4 h, beginning 2 h into the light cycle. Poloxamer 407 (Merck, Darmstadt, Germany) diluted in saline was intraperitoneally injected at a dose of 1 mg/g body weight to inhibit lipoprotein lipase (LPL) activity. Blood was collected from the tail vein into capillary blood collection tubes before injection and at 1, 2, 4, and 8 h postinjection. Serum was isolated from blood and assayed for TG using a colorimetric kit.

Intestinal lipid absorption and secretion

Overnight-unfed mice (~15 h) were intraperitoneally injected with poloxamer 407 diluted in saline at a dose of 1 mg/g body weight to inhibit LPL activity. After 1 h, mice were orally gavaged with 20% emulsified intralipid (10 μl/g body weight). Blood was collected from the tail vein into capillary blood collection tubes before gavage and at 1, 2, 3, and 4 h postgavage. Serum was isolated from blood samples and assayed for TG using a colorimetric kit.

Blood and tissue collection

Mice were anesthetized with isoflurane, and blood was collected from the retro-orbital plexus through heparin-coated capillary tubes (Thermo Fisher Scientific). Blood samples were allowed to clot on ice and were then centrifuged for 10 min at 10,000 g. Separated serum was divided into aliquots and stored at −80°C. Organs were excised, weighed, and flash-frozen in liquid nitrogen before being stored at −80°C.

Complete blood count analysis

A complete blood count on blood samples was performed at the Pathology Phenotyping Core at the Johns Hopkins University School of Medicine. Blood samples were collected from the femoral vein using EDTA-coated blood collection tubes (Sarstedt) and analyzed using Procyte Dx analyzer (Idexx Laboratories, Westbrook, ME, USA).

Serum lipoprotein analysis

Lipoprotein analyses were performed at the Metabolism core at the Baylor College of Medicine (Houston, TX, USA). Serum (300 μl) was pooled from 10 mice of each genotype (30 μl/mouse). Pooled serum samples from WT and KO mice were subjected to fractionation using FPLC followed by quantification of TG and cholesterol levels in each fraction.

Heparin releasable LPL activity in serum

Mice were unfed overnight (~13 h) and then given free access to food for 2 h. The experiment was conducted as previously described by Qiao et al. (64). Mice were briefly anesthetized with isoflurane and retro-orbitally injected with heparin sodium salt (MilliporeSigma) diluted in saline at a dose of 300 U/kg body weight. Blood was collected from the tail vein into capillary blood collection tubes before injection and 5 min postinjection. Serum was isolated from blood samples. Preheparin serum was used to measure serum TG levels. Total lipase activity was measured using a commercial kit (BioVision, Milpitas, CA, USA) in postheparin serum.

Mouse histology

Adipose and liver tissues from littermate-matched WT and myonectin-KO mice were fixed in 10% formalin at 4°C overnight. Fixed tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) at the Histology Reference Laboratory at the Johns Hopkins University School of Medicine.

Adipocyte cell size analysis

Adipocyte size was measured using the MRI Adipocyte Tools macro in the ImageJ software [National Institutes of Health (NIH), Bethesda, MD, USA]. All cells in 1 field of view at ×100 magnification per tissue section per mouse were analyzed from a total of 5 WT and 5 KO littermate-matched mice for visceral (gonadal) white adipose tissue (WAT) and 4 WT and 5 KO littermate-matched mice for subcutaneous inguinal WAT (iWAT). On average, 230 cells were measured for each sample.

Extraction and quantification of mouse hepatic lipid contents

A total of 50–80 mg of frozen liver tissue was homogenized in 500 μl ultra-pure water. A total of 200 μl lysate was transferred to a new tube, and 1 ml of 2:1 chloroform:methanol was added. After thorough mixing, samples were spun at 1700 rpm for 5 min at 4°C. The lower chloroform phase was transferred to a new tube and dried in a speed vacuum. Extracted lipids were resuspended in 5% Triton X-100 in saline. Samples were assayed for TG and cholesterol using commercially available colorimetric kits (Thermo Fisher Scientific). Protein content was measured in the original water lysate using a bicinchoninic acid assay (Thermo Fisher Scientific), and lipid levels were normalized to total protein.

Western blotting

Frozen tissue samples were homogenized in RIPA buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% Triton X-100; 0.25% deoxycholate) supplemented with a protease inhibitor cocktail (MilliporeSigma) and a phosphatase inhibitor cocktail (Roche, Basel, Switzerland). Protein content was measured using a bicinchoninic acid assay. Samples for Western blotting were diluted in SDS loading dye (final concentration: 50 mM Tris-HCl, pH 7.4, 2% SDS, 6% glycerol, 1% 2-ME, and 0.01% bromphenol blue) and denatured at 95°C for 10 min. A total of 10–20 μg total protein was loaded into each lane of a 10% polyacrylamide gel (Bio-Rad, Hercules, CA, USA) and separated by electrophoresis. The Bio-Rad Trans-Blot Turbo semidry system was used to transfer protein onto PVDF membranes. Blots were blocked in 5% nonfat milk in PBS with Tween 20 (PBST) and exposed to primary antibodies overnight at 4°C. After washing, blots were exposed to secondary antibodies conjugated to horseradish peroxidase (HRP) for 1 h and developed in ECL (Amersham ECL Select; GE Healthcare, Chicago, IL, USA). Bands were visualized with MultiImage III FluorChem Q (Alpha Innotech, San Leandro, CA, USA) and quantified using Alphaview Software (Alpha Innotech). All primary antibodies were diluted 1:1000 in PBST + 0.02% sodium azide, and all secondary antibodies were diluted 1:5000 in 5% nonfat milk in PBST. Antibodies used: LPL (GTX101125; GeneTex, Irvine, CA, USA), heat shock cognate 71 kDa protein (Hsc70) (sc-7298; Santa Cruz Biotechnology, Dallas, TX, USA), perilipin 2 (PA1-16972; Thermo Fisher Scientific), mouse IgG-HRP (7076; Cell Signaling Technology, Danvers, MA, USA), and rabbit IgG-HRP (7074; Cell Signaling Technology).

Liver metabolomics analysis

Metabolite quantifications of WT (n = 10) and myonectin-KO (n = 10) liver samples were performed by Metabolon (Morrisville, NC, USA) using the metabolomics platform. Sample processing, compound identification by tandem mass spectrometry, metabolite quantification and data normalization, and data extraction and curation were all performed according to the manufacturer’s protocol.

Stimulated adipose tissue lipolysis

Mice were unfed for 5 h beginning at 9:30 am. The β₃-adrenergic receptor agonist, CL 316,243 (Santa Cruz Biotechnology), was diluted in saline and intraperitoneally injected at a dose of 1 mg/kg body weight. Blood was collected from the tail vein into capillary blood collection tubes before injection and at 15 min postinjection. Blood glucose was monitored with a glucometer before and 15 min after CL 316,243 injection. Serum was separated from blood and assayed for NEFAs and glycerol using calorimetric kits (Fujifilm and MilliporeSigma, respectively).

Tissue LPL activity

Tissue LPL activity was measured as previously described by Mizunoya et al. (65). Briefly, frozen tissue samples were pulverized in liquid nitrogen using a mortar and pestle. A total of ~50 mg pulverized tissue was incubated in Krebs-Ringer solution containing bovine serum albumin and heparin. After clarifying, the supernatant was used to measure LPL activity using a commercial kit (Roar Biomedical, New York, NY, USA). Activity was normalized to tissue weight.

Real-time quantitative PCR analysis

RNA was isolated from tissues using Trizol reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol and treated with DNAse I (New England Biolabs, Ipswich, MA, USA) to remove genomic DNA. For each sample, 1–2 μg total RNA was reverse transcribed using random primers and the GoScript reverse transcription system from Promega (Madison, WI, USA). Real-time quantitative PCR was performed on a CFX connect system using iTaq Universal SYBR Green PCR master mix (Bio-Rad). Gene expression was first normalized to 36b4 (also known as acidic ribosomal phosphoprotein P0) to generate a ΔC_t value, and ΔΔC_t was obtained by normalizing data to mean ΔC_t of the control group (66). Information on primers used are available upon request.

Maximal exercise capacity

Maximal sprint and endurance exercise tests were performed as we previously described (67). Briefly, 12-wk-old mice fed a standard chow diet were first subjected to the endurance exercise test. Mice were acclimated to the treadmill for 3 d before the experiment, and the exercise test was carried out on the fourth day. Blood glucose and lactate values were measured before and after exercise using glucose and lactate meters (Nova Biomedical). Blood was collected from the tail vein into capillary blood collection tubes before and after exercise, and silver nitrate sticks were used to cauterize the wound. Mice were run in multiple groups, beginning at 9:30 am until exhaustion. After 2.5 wk of recovery, mice were subjected to the sprint exercise test. At 1 d before the experiment, mice were reacclimated to the treadmill using the same protocol as the original d-3 acclimation. For the sprint exercise experiment, mice were run in multiple groups, beginning at 8:30 am until exhaustion. As with the endurance test, blood, glucose, and lactate were collected before and after exercise. The experimenter was blinded with respect to genotype during all exercise tests.

Recovery after endurance exercise

Mice used in this experiment were fed a standard chow diet. Mice were acclimated to the treadmill as previously described, but with the addition of a fourth acclimation day in which mice ran on the treadmill at 14 m/min for 5 min at a 10° incline (67). The endurance exercise run was performed on the fifth day, when the mice were 11 wk of age. Mice were unfed for 2 h in the morning before exercise. The run was conducted as previously described by Leick et al. (68): 60 min at a speed of 14 m/min at a 10° incline. A shock grid at the back of the treadmill encouraged mice to continue running by administering a mild shock each time they stepped off the belt. If any mice met the predetermined criteria for exhaustion before the end of the run, they were removed from the treadmill and their data were omitted from analysis (67). The experimenter was blinded with respect to genotype during the treadmill run. Blood glucose and lactate values were measured at the indicated time points using glucose and lactate meters (Nova Biomedical). A single cohort of mice received a glucose gavage within 5 min of the end of the run (0.5 mg glucose/g body weight dissolved in saline). All mice were allowed to recover in their cages with free access to water but no food. After 2 h of recovery following the end of the run, mice were euthanized. Blood and tissues were collected as described above.

Tissue glycogen measurement

Tissue glycogen was measured using a protocol adapted from the NIH-funded Mouse Metabolic Phenotyping Centers (https://mmpc.org/shared/protocols.aspx). Frozen whole liver and gastrocnemius muscle were pulverized in liquid nitrogen. A total of ~25 mg liver tissue or 50 mg muscle were homogenized in 400 μl 0.9 N perchloric acid. Samples were clarified by centrifuging at 10,000 g for 10 min at 4°C and transferring the supernatant to a new tube. Supernatant (50 μl) was transferred to each of 2 reaction tubes, along with 25 μl 1 M potassium bicarbonate and 125 μl 0.4 M sodium acetate buffer (pH 4.8) with or without 5 mg/ml amyloglucosidase (MilliporeSigma). Reactions were incubated for 2 h at 40°C on a heat block. After 2 h, reactions were neutralized with NaOH and then vortexed and centrifuged for 3 min at 10,000 g at room temperature. A total of 20 μl of supernatant from each reaction was used to measure glucose using an Amplex Red glucose assay kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Amyloglucosidase hydrolyzes glycogen into free glucose monomers. For each tissue sample, the amount of glucose in the condition without amyloglucosidase (measuring free glucose) was subtracted from the amount of glucose in the condition with amyloglucosidase (measuring free glucose + glucose from glycogen) to yield the amount of glucose from glycogen only. This value was then normalized to tissue weight.

Myonectin is not required for the physiologic response to exercise

Under basal conditions, myonectin is primarily produced in skeletal muscle, and its expression can be further induced by exercise (28, 29). We therefore first tested whether it is required for the physiologic response to exercise. During both maximal sprint and endurance exercise on a treadmill, loss of myonectin had no impact on total running distance and the postexercise serum levels of glucose, lactate, NEFA, TG, and ketone (β-hydroxybutyrate) in female WT and KO mice (Table 1). However, we did observe significantly higher pre-exercise serum ketones (β-hydroxybutyrate) in KO female mice relative to WT littermate controls. For male mice, with the exception of postsprint exercise serum TG levels that were significantly lower in the KO animals, no significant genotypic differences were noted in exercise parameters (Table 1). In the postexercise recovery phase (with or without glucose gavage postexercise), myonectin deficiency did not significantly affect the ability of KO animals to replenish their skeletal muscle or liver glycogen stores (Table 2). These results indicate that myonectin is not required for physiologic response and adaptation to an acute bout of exercise.

TABLE 1

Time to exhaustion, total distance run, and blood parameters during an endurance exercise capacity test or a sprint exercise capacity test in chow-fed male and female WT and myonectin-KO mice

Parameter	Female mice			Male mice
	WT	KO	P	WT	KO	P
Endurance exercise capacity
N	14	15		9	14
Time to exhaustion (min)	116.1 ± 5.253	125.0 ± 4.317	0.2052	99.1 ± 7.172	101.5 ± 3.694	0.7714
Running distance (m)	1770 ± 117.5	1968 ± 101.6	0.2135	1406 ± 141.2	1447 ± 75.8	0.8031
Pre-exercise glucose (mg/dl)	161.9 ± 4.655	157.9 ± 3.933	0.5253	190.2 ± 6.357	183.3 ± 3.758	0.3640
Postexercise glucose (mg/dl)	148.5 ± 11.27	128.9 ± 6.84	0.1508	175.1 ± 17.39	161.4 ± 8.85	0.4953
Pre-exercise lactate (mM)	2.000 ± 0.0989	1.873 ± 0.1197	0.4219	1.433 ± 0.1424	1.550 ± 0.1763	0.6120
Postexercise lactate (mM)	5.232 ± 0.3179	5.013 ± 0.3570	0.6508	5.300 ± 0.6388	4.779 ± 0.4960	0.5278
Pre-exercise TG (mg/dl)	56.75 ± 5.872	50.17 ± 4.273	0.3740	53.01 ± 7.726	57.32 ± 6.143	0.6675
Postexercise TG (mg/dl)	52.00 ± 2.629	53.31 ± 2.485	0.7202	37.13 ± 2.472	43.26 ± 2.363	0.0905
Pre-exercise NEFA (mM)	1.225 ± 0.0940	1.035 ± 0.0652	0.1113	1.196 ± 0.0543	1.172 ± 0.1284	0.8662
Postexercise NEFA (mM)	2.387 ± 0.1764	2.756 ± 0.1466	0.1196	1.633 ± 0.1979	2.020 ± 0.2182	0.2049
Pre-exercise β-hydroxybutyrate (μM)	122.4 ± 8.09*	163.8 ± 17.07*	0.0405*	139.2 ± 10.93	133.0 ± 15.28	0.7445
Postexercise β-hydroxybutyrate (μM)	545.7 ± 47.10	623.3 ± 52.92	0.2830	377.0 ± 58.62	370.2 ± 44.52	0.9278
Sprint exercise capacity
N	15	15		9	12
Time to exhaustion (min)	14.52 ± 0.4734	14.71 ± 0.5862	0.7999	12.90 ± 0.6590	12.97 ± 0.4013	0.9373
Running distance (m)	244.4 ± 13.20	250.6 ± 17.11	0.7764	200.8 ± 17.31	201.2 ± 10.79	0.9851
Pre-exercise glucose (mg/dl)	169.6 ± 5.266	176.1 ± 4.392	0.3491	180.8 ± 4.481	186.1 ± 5.140	0.4461
Postexercise glucose (mg/dl)	180.8 ± 3.558	176.3 ± 5.092	0.4788	221.9 ± 8.093	209.8 ± 5.042	0.2219
Pre-exercise lactate (mM)	2.270 ± 0.2750	2.027 ± 0.1584	0.4512	1.711 ± 0.1184	1.917 ± 0.1753	0.3440
Postexercise lactate (mM)	11.02 ± 0.4419	10.29 ± 0.5822	0.3293	8.872 ± 1.116	9.700 ± 0.803	0.5559
Pre-exercise TG (mg/dl)	48.40 ± 4.332	49.07 ± 4.985	0.9203	59.03 ± 4.007	54.92 ± 3.592	0.4559
Postexercise TG (mg/dl)	46.93 ± 2.458	48.55 ± 2.033	0.6159	56.55 ± 3.604*	45.31 ± 2.351*	0.0202*
Pre-exercise NEFA (mM)	1.083 ± 0.1190	0.856 ± 0.1453	0.2368	1.199 ± 0.0983	1.307 ± 0.1314	0.5187
Postexercise NEFA (mM)	1.012 ± 0.0526	0.919 ± 0.1231	0.4964	0.982 ± 0.1055	0.814 ± 0.0894	0.2403
Pre-exercise β-hydroxybutyrate (μM)	199.6 ± 13.41*	244.9 ± 15.74*	0.0371*	161.9 ± 21.62	203.9 ± 33.47	0.3055
Postexercise β-hydroxybutyrate (μM)	224.0 ± 17.87	239.6 ± 14.98	0.5103	204.8 ± 24.29	224.4 ± 20.16	0.5423

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Mice were 12 wk of age for the endurance test and 14.5 wk of age for the sprint test. *P < 0.05.

TABLE 2

Chow-fed male and female WT and myonectin-KO mice (at 11 wk of age) were subjected to an endurance run on the treadmill, and blood glucose and lactate values were measured at the indicated time points

Parameter	Female mice			Male mice
	WT	KO	P	WT	KO	P
No glucose gavage postexercise
N	6	5		5	6
Body weight (g)	18.23 ± 0.3836	18.10 ± 0.8701	0.8934	25.50 ± 0.7668	24.38 ± 0.7631	0.3293
Liver weight (g)	0.7950 ± 0.0193	0.7340 ± 0.0370	0.1928	1.076 ± 0.0367	1.040 ± 0.0357	0.4999
Pre-exercise glucose (mg/dl)	181.5 ± 5.30	187.0 ± 10.70	0.6614	211.8 ± 6.63	201.0 ± 8.67	0.3487
0 min postexercise glucose (mg/dl)	184.5 ± 6.35	191.6 ± 13.31	0.6478	207.0 ± 13.02	211.3 ± 11.13	0.8064
30 min postexercise glucose (mg/dl)	158.2 ± 7.85	150.4 ± 11.60	0.5959	159.0 ± 6.46	158.0 ± 6.81	0.9175
60 min postexercise glucose (mg/dl)	144.7 ± 13.11	154.8 ± 6.84	0.5139	140.8 ± 10.96	163.3 ± 5.90	0.1183
120 min postexercise glucose (mg/dl)	127.8 ± 5.88	135.2 ± 5.08	0.3682	145.4 ± 12.84	154.2 ± 4.74	0.5496
Pre-exercise lactate (mM)	1.750 ± 0.1057	1.560 ± 0.0980	0.2200	2.400 ± 0.4572	2.033 ± 0.1333	0.4783
0 min postexercise lactate (mM)	3.950 ± 0.2814	4.300 ± 0.7987	0.6965	4.460 ± 0.1778	4.400 ± 0.3907	0.8928
Muscle glycogen (nmol glucose from glycogen/mg tissue)	9.75 ± 0.5824	10.53 ± 1.3198	0.6115	11.16 ± 1.085	12.37 ± 1.981	0.6097
Liver glycogen (nmol glucose from glycogen/mg tissue)	30.81 ± 7.6927	12.10 ± 0.8849	0.0591	67.37 ± 19.70	78.24 ± 11.16	0.6470
With glucose gavage postexercise
N	5	6		5	5
Body weight (g)	19.94 ± 0.3444	20.33 ± 0.8887	0.6932	24.12 ± 0.5417	24.86 ± 0.6823	0.4216
Liver weight (g)	0.8300 ± 0.0319	0.8417 ± 0.0464	0.8409	1.006 ± 0.0279	1.008 ± 0.0323	0.9638
Pre-exercise glucose (mg/dl)	162.2 ± 7.51	160.7 ± 4.82	0.8684	198.2 ± 7.37	192.2 ± 5.21	0.5270
0 min postexercise glucose (mg/dl)	150.8 ± 17.89	159.5 ± 8.31	0.6754	197.4 ± 12.48	192.0 ± 10.22	0.7467
30 min postexercise glucose (mg/dl)	137.6 ± 9.48	153.2 ± 4.52	0.1906	179.0 ± 2.19	172.6 ± 8.35	0.4948
60 min postexercise glucose (mg/dl)	127.7 ± 6.74	129.0 ± 2.00	0.8649	156.0 ± 13.00	154.7 ± 0.67	0.9350
120 min postexercise glucose (mg/dl)	103.8 ± 12.01	105.5 ± 7.98	0.9094	139.4 ± 7.90	137.8 ± 4.42	0.8652
Pre-exercise lactate (mM)	1.580 ± 0.1068	1.517 ± 0.0601	0.6225	1.540 ± 0.0872	1.740 ± 0.1568	0.3060
0 min postexercise lactate (mM)	6.520 ± 1.3782	4.433 ± 0.7697	0.2316	2.800 ± 0.1140	4.600 ± 1.1406	0.1901
Muscle glycogen (nmol glucose from glycogen/mg tissue)	9.63 ± 0.5762	10.56 ± 0.2763	0.1976	12.15 ± 0.835	12.91 ± 0.836	0.5347
Liver glycogen (nmol glucose from glycogen/mg tissue)	27.91 ± 4.6121	44.47 ± 9.4856	0.1596	42.61 ± 17.04	47.36 ± 11.02	0.8216

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Half of the mice received a glucose gavage immediately after completing the run, and mice were allowed to recover for 2 h before euthanization.

Myonectin-KO mice fed a control LFD show no overt metabolic phenotypes

Given its potential role as a postprandial metabolic regulator (28, 36), we tested whether myonectin is necessary for metabolic homeostasis under basal conditions. Male and female WT and KO mice were fed a control LFD for 12 wk, beginning at 6 wk of age. Neither sex displayed genotypic differences in body weight over time or in body composition (Supplemental Fig. S1A, B). Organ weights (heart, kidney, spleen, liver, and adipose tissue) were also not different between genotypes of either sex (Supplemental Table S1). Indirect calorimetry revealed no differences in ad libitum food intake, metabolic rate, or EE between WT and KO mice of either sex (Table 3). However, ad libitum fed female KO mice had a higher RER in the dark cycle and lower physical activity in the light cycle. Similar to the female KO mice, ad libitum fed male KO mice also had significantly lower physical activity in the light cycle and over a 24-h period. Myonectin is robustly induced upon refeeding following an unfed period (36). We therefore challenged the WT and KO mice with a 24-h unfed period followed by 24 h of free access to food while they were in the metabolic cages. No genotypic differences in either sex were observed during the unfed period (Table 3). However, in the refeeding phase, female KO mice had lower RER in the light cycle, and male KO mice had significantly reduced physical activity in the dark cycle and over a 24-h period (Table 3). Because myonectin is also up-regulated after an acute bout of exercise (28), we provided the WT and KO mice with a voluntary running wheel in the metabolic cages in a separate experiment. No major genotypic differences in either sex were observed (Supplemental Table S2).

TABLE 3.

Indirect calorimetry, physical activity, and food intake analysis of male and female mice fed a control LFD

Parameter	Dark cycle			Light cycle			24 h
	WT	KO	P	WT	KO	P	WT	KO	P
LFD (female)
Ad libitum
N	6	6		6	6		6	6
Body weight (g)							23.69 ± 0.71	23.67 ± 0.78	0.9816
Food intake (kcal)	11.34 ± 0.5525	13.30 ± 0.8616	0.0979	4.00 ± 0.3027	3.52 ± 0.4833	0.4293	15.34 ± 0.6757	16.98 ± 0.7156	0.1262
Vo2 (ml/kg lean mass/h)	6226 ± 360.15	6211 ± 266.79	0.9751	5078 ± 221.79	5153 ± 47.77	0.7562	5650 ± 239.43	5680 ± 131.95	0.9144
Vco2 (ml/kg lean mass/h)	5980 ± 288.22	6222 ± 229.13	0.5264	4337 ± 213.50	4453 ± 68.20	0.6219	5153 ± 193.94	5333 ± 91.15	0.4282
RER (Vco2/Vo2)	0.9632 ± 0.0100*	1.0035 ± 0.0139*	0.0424*	0.8533 ± 0.0119	0.8644 ± 0.0120	0.5242	0.9133 ± 0.0087	0.9398 ± 0.0101	0.0750
EE (kcal/kg lean mass/h)	31.12 ± 1.7285	31.36 ± 1.2895	0.9125	24.72 ± 1.1017	25.14 ± 0.2314	0.7194	27.90 ± 1.1485	28.24 ± 0.6070	0.8019
Total activity (beam breaks)	89,169 ± 22,955	76,618 ± 13,112	0.6477	27,651 ± 1674*	20,527 ± 1654*	0.0128*	116,819 ± 24,438	97,145 ± 13,992	0.5047
Ambulatory activity (counts)	62,096 ± 18,035	51,144 ± 8978	0.6028	17,165 ± 1826*	11,441 ± 1462*	0.0355*	79,261 ± 19,212	62,585 ± 9459	0.4607
Unfed
N	5	6		5	6		5	6
Body weight (g)							20.68 ± 0.84	20.50 ± 0.90	0.8808
Food intake (kcal)	0	0		0	0		0	0
Vo2 (ml/kg lean mass/h)	5658 ± 165.67	5958 ± 312.73	0.4239	4020 ± 259.17	4275 ± 335.13	0.5610	4837 ± 208.50	5108 ± 296.49	0.4736
Vco2 (ml/kg lean mass/h)	4219 ± 141.64	4365 ± 201.45	0.5683	3000 ± 211.40	3130 ± 221.65	0.6798	3607 ± 172.87	3742 ± 196.15	0.6204
RER (Vco2/Vo2)	0.7453 ± 0.0074	0.7341 ± 0.0063	0.2791	0.7451 ± 0.0097	0.7342 ± 0.0118	0.4923	0.7453 ± 0.0078	0.7336 ± 0.0067	0.2872
EE (kcal/kg lean mass/h)	26.78 ± 0.8006	28.11 ± 1.4406	0.4467	19.03 ± 1.2461	20.17 ± 1.5478	0.5812	22.90 ± 1.0048	24.10 ± 1.3710	0.4981
Total activity (beam breaks)	96,004 ± 13,580	92,982 ± 17,340	0.8939	46,965 ± 11,932	48,531 ± 22,673	0.9529	142,969 ± 22,646	141,513 ± 35,927	0.9735
Ambulatory activity (counts)	68,721 ± 9475	67,799 ± 12,992	0.9556	32,873 ± 8610	34,541 ± 17,317	0.9336	101,594 ± 15,363	102,340 ± 26,904	0.9814
Refeed
N	5	6		5	6		5	6
Body weight (g)							23.17 ± 1.06	22.45 ± 0.79	0.6028
Food intake (kcal)	14.65 ± 0.7735	15.42 ± 2.0633	0.7432	5.50 ± 0.1779	5.13 ± 0.4825	0.5040	20.15 ± 0.9101	20.19 ± 1.6992	0.9856
Vo2 (ml/kg lean mass/h)	5347 ± 222.17	5673 ± 110.57	0.2376	4839 ± 148.65	4978 ± 104.46	0.4699	5102 ± 182.80	5340 ± 51.57	0.2694
Vco2 (ml/kg lean mass/h)	5408 ± 213.09	5666 ± 131.89	0.3373	5000 ± 148.67	4944 ± 140.85	0.7922	5211 ± 179.39	5321 ± 53.04	0.5836
RER (Vco2/Vo2)	1.0119 ± 0.0089	0.9986 ± 0.0087	0.3115	1.0338 ± 0.0145*	0.9925 ± 0.0087*	0.0463*	1.0219 ± 0.0110	0.9965 ± 0.0043	0.0825
EE (kcal/kg lean mass/h)	27.06 ± 1.1056	28.62 ± 0.5768	0.2562	24.62 ± 0.7348	25.08 ± 0.5710	0.6353	25.89 ± 0.9105	26.93 ± 0.2572	0.3237
Total activity (beam breaks)	38,270 ± 3078	43,979 ± 4778	0.3438	16,763 ± 1463	14,686 ± 1536	0.3532	55,032 ± 4218	58,665 ± 5862	0.6276
Ambulatory activity (counts)	23,748 ± 4240	27,082 ± 2675	0.5276	8759 ± 1978	7406 ± 831	0.5538	32,507 ± 6124	34,487 ± 3267	0.7848
LFD (male)
Ad libitum
N	6	6		6	6		6	6
Body weight (g)							33.78 ± 0.93	32.27 ± 1.54	0.4267
Food intake (kcal)	12.10 ± 0.4483	12.19 ± 1.0683	0.9447	4.68 ± 0.5210	4.32 ± 0.2848	0.5692	16.78 ± 0.9393	16.51 ± 1.2516	0.8670
Vo2 (ml/kg lean mass/h)	5140 ± 336.81	4764 ± 122.47	0.3327	4507 ± 317.60	4266 ± 87.28	0.4925	4821 ± 327.53	4512 ± 93.37	0.3996
Vco2 (ml/kg lean mass/h)	4979 ± 325.83	4702 ± 169.58	0.4737	4037 ± 276.07	3855 ± 106.02	0.5594	4506 ± 300.88	4274 ± 130.21	0.5038
RER (Vco2/Vo2)	0.9691 ± 0.0099	0.9857 ± 0.0141	0.3607	0.8976 ± 0.0157	0.9032 ± 0.0093	0.7630	0.9355 ± 0.0120	0.9463 ± 0.0111	0.5245
EE (kcal/kg lean mass/h)	25.74 ± 1.6833	23.97 ± 0.6710	0.3620	22.17 ± 1.5447	21.02 ± 0.4594	0.5046	23.94 ± 1.6160	22.48 ± 0.5144	0.4205
Total activity (beam breaks)	31,086 ± 2573	25,107 ± 2927	0.1565	14,819 ± 1041*	10,042 ± 1007*	0.0080*	45,905 ± 2828*	35,148 ± 3672*	0.0443*
Ambulatory activity (counts)	15,473 ± 1901	12,719 ± 1800	0.3176	6390 ± 910	4123 ± 528	0.0631	21,862 ± 2305	16,841 ± 2167	0.1436
Unfed
N	6	6		6	6		6	6
Body weight (g)							30.40 ± 1.00	29.26 ± 1.54	0.5519
Food intake (kcal)	0	0		0	0		0	0
Vo2 (ml/kg lean mass/h)	4821 ± 351.50	4499 ± 137.61	0.4240	3773 ± 252.60	3497 ± 141.25	0.3683	4292 ± 299.08	3996 ± 125.83	0.3935
Vco2 (ml/kg lean mass/h)	3560 ± 273.93	3319 ± 101.56	0.4404	2715 ± 185.86	2508 ± 110.25	0.3650	3133 ± 227.98	2912 ± 97.03	0.4025
RER (Vco2/Vo2)	0.7376 ± 0.0052	0.7377 ± 0.0017	0.9839	0.7192 ± 0.0037	0.7165 ± 0.0058	0.7110	0.7294 ± 0.0037	0.7284 ± 0.0029	0.8459
EE (kcal/kg lean mass/h)	22.78 ± 1.6772	21.25 ± 0.6499	0.4268	17.74 ± 1.1923	16.43 ± 0.6730	0.3673	20.23 ± 1.4214	18.83 ± 0.5989	0.3950
Total activity (beam breaks)	46,032 ± 3459	39,156 ± 4027	0.2250	14,328 ± 1523	12,284 ± 1298	0.3317	60,360 ± 3607	51,439 ± 4403	0.1493
Ambulatory activity (counts)	26,838 ± 2492	22,431 ± 2843	0.2713	6571 ± 1026	5685 ± 803	0.5127	33,409 ± 2625	28,116 ± 3097	0.2223
Refeed
N	6	6		6	6		6	6
Body weight (g)							32.65 ± 1.00	31.51 ± 1.23	0.4913
Food intake (kcal)	16.11 ± 0.7839	15.39 ± 0.7509	0.5229	4.01 ± 0.2276	3.91 ± 0.1932	0.7383	20.12 ± 0.9052	19.29 ± 0.8238	0.5175
Vo2 (ml/kg lean mass/h)	5289 ± 306.05	4745 ± 95.61	0.1413	4366 ± 278.17	4085 ± 89.06	0.3716	4848 ± 289.28	4430 ± 90.21	0.2167
Vco2 (ml/kg lean mass/h)	5092 ± 280.39	4618 ± 120.98	0.1657	4179 ± 242.68	3952 ± 104.51	0.4205	4655 ± 257.94	4300 ± 105.02	0.2446
RER (Vco2/Vo2)	0.9637 ± 0.0067	0.9725 ± 0.0088	0.4410	0.9595 ± 0.0155	0.9675 ± 0.0137	0.7086	0.9618 ± 0.0099	0.9704 ± 0.0089	0.5320
EE (kcal/kg lean mass/h)	26.45 ± 1.5117	23.79 ± 0.5101	0.1458	21.81 ± 1.3535	20.45 ± 0.4541	0.3787	24.23 ± 1.4185	22.20 ± 0.4674	0.2214
Total activity (beam breaks)	33,740 ± 2923*	23,819 ± 1962*	0.0207*	10,661 ± 986	8470 ± 891	0.1307	44,401 ± 3607*	32,289 ± 2359*	0.0212*
Ambulatory activity (counts)	15,749 ± 1880	11,041 ± 1046	0.0608	4111 ± 452	3088 ± 449	0.1393	19,860 ± 2129*	14,129 ± 1240*	0.0483*

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At the time of the study, LFD-fed mice were 27 wk of age. LFD-fed mice were given 2 d to acclimate to the metabolic cages. Ad libitum data were collected on the third day. Mice were denied access to food but had free access to water on d 4 (unfed data), and refeed data were collected on d 5 when mice regained free access to food. *P < 0.05.

To assess whether myonectin deficiency alters carbohydrate metabolism, glucose tolerance tests (GTTs) were performed. The rate of glucose uptake in peripheral tissues in response to glucose injection was not different between genotypes of either sex (Supplemental Fig. S1C, D). The magnitude of insulin secretion in response to a rise in blood glucose during GTT did not differ between genotypes in males (Supplemental Fig. S1E). We also performed ITTs to assess insulin action in peripheral tissue. The rate of glucose disposal in response to a bolus of insulin did not differ between genotypes of either sex (Supplemental Fig. S1F, G). We previously showed that recombinant myonectin infusion promotes lipid uptake in mice (28). To test whether myonectin deficiency affects the ability of mice to handle an acute lipid load, we performed lipid tolerance tests. Overnight-unfed mice were orally gavaged with a bolus of emulsified intralipid, and serum lipid levels were measured over time to assess the rate of lipid uptake and clearance by peripheral tissues. No genotypic differences were observed in the rate of clearance of serum TG or NEFA (Supplemental Fig. S1H, I). Aside from physical activity in the ad libitum fed state or during refeeding, our data indicate that myonectin is dispensable for whole-body carbohydrate and lipid metabolism under basal conditions.

Minimal impact of myonectin deficiency on carbohydrate metabolism in the context of obesity

We next determined whether myonectin is required for a proper response to metabolic stress. A separate cohort of male and female WT and KO mice was challenged with an HFD, beginning at 5 wk of age, to induce obesity and insulin resistance. We performed the same sets of metabolic tests as in the control LFD-fed cohorts to determine whether myonectin deficiency exacerbates or improves the pathophysiological phenotypes associated with diet-induced obesity and insulin resistance. Body weight and body composition did not differ between genotypes of either sex (Fig. 2A, B). Despite no difference in overall fat mass, the gonadal fat pad was larger in myonectin-KO female mice relative to WT littermates (Supplemental Table S1). Indirect calorimetry analyses indicated no differences in metabolic rate, physical activity, or EE between genotypes of either sex under ad libitum fed conditions or during a 24-h unfed period and 24-h refeed period (Table 4). However, we noted that ad libitum fed female KO mice had significantly reduced food intake in the light cycle. In the refeeding phase following an unfed period, female KO mice had reduced food intake and lower RER in the dark cycle; a reduction in food intake was also observed over the 24-h refeed period. In contrast to female mice, the only difference seen in KO male mice was reduced RER in the light cycle in the ad libitum fed state (Table 4). Myonectin-deficient female mice also showed a modest impairment in the rate of glucose uptake in peripheral tissues in response to glucose or insulin injection (Fig. 2C, F). Myonectin-deficient male mice, however, were indistinguishable from WT littermates in GTTs and ITTs (Fig. 2D, G). The magnitude of insulin secretion in response to glucose injection during the GTT did not differ between genotypes in males (Fig. 2E). Together, these results indicate that myonectin deficiency has minimal impact on whole-body carbohydrate metabolism in the pathophysiological context of diet-induced obesity.

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Figure 2

There are no overt carbohydrate metabolism phenotypes in the myonectin-KO mice fed an HFD. A) Body weights of female (WT, n = 8; KO, n = 8) and male (WT, n = 12–13; KO, n = 16) mice over time. Mice were weaned at 3.5 wk of age onto a standard chow diet. At 5 wk of age, the diet was switched to an HFD. B) Body composition analysis of female (WT, n = 7; KO, n = 8) and male (WT, n = 12; KO, n = 16) mice at 28 wk of age. C) Blood glucose levels during intraperitoneal GTTs in 18-wk-old female mice (WT, n = 8; KO, n = 8). D) Blood glucose levels during oral GTT in 19-wk-old male mice (WT, n = 12; KO, n = 12). E) Serum insulin levels in male mice at 0 min before glucose gavage (WT, n = 11; KO, n = 9) and at 15 min after glucose gavage (WT, n = 12; KO, n = 11); for some samples, insulin levels were below the threshold of detection. F, G) Blood glucose levels during ITTs in female mice (WT, n = 7; KO, n = 8) and male mice (WT, n = 11; KO, n = 15) at 23 wk of age. Insulin was injected at a dose of 1.2 U/kg body weight for female mice and 1.5 U/kg body weight for male mice. *P < 0.05.

TABLE 4.

Indirect calorimetry, physical activity, and food intake analysis of male and female mice fed an HFD

Parameter	Dark cycle			Light cycle			24 h
	WT	KO	P	WT	KO	P	WT	KO	P
HFD (female)
Ad libitum
N	6	6		6	6		6	6
Body weight (g)							39.26 ± 2.16	39.25 ± 2.03	0.9978
Food intake (kcal)	7.78 ± 0.936	6.02 ± 1.133	0.2629	2.77 ± 0.278*	1.70 ± 0.346*	0.0419*	10.55 ± 1.118	7.71 ± 1.404	0.1522
Vo2 (ml/kg lean mass/h)	5829 ± 241.71	5891 ± 32.68	0.8086	5275 ± 214.59	5244 ± 153.42	0.9072	5550 ± 225.09	5567 ± 74.91	0.9464
Vco2 (ml/kg lean mass/h)	4417 ± 174.41	4366 ± 30.95	0.7821	3981 ± 126.93	3886 ± 103.03	0.5756	4198 ± 147.35	4126 ± 52.17	0.6601
RER (Vco2/Vo2)	0.7584 ± 0.0105	0.7412 ± 0.0070	0.2064	0.7561 ± 0.0082	0.7415 ± 0.0046	0.1591	0.7573 ± 0.0088	0.7412 ± 0.0055	0.1582
EE (kcal/kg lean mass/h)	27.68 ± 1.1285	27.85 ± 0.1266	0.8840	25.03 ± 0.9735	24.79 ± 0.7097	0.8482	26.35 ± 1.0355	26.32 ± 0.3409	0.9822
Total activity (beam breaks)	26,652 ± 5042	30,357 ± 4571	0.5983	13,739 ± 2849	14,363 ± 2550	0.8736	40,391 ± 7665	44,720 ± 6227	0.6709
Ambulatory activity (counts)	12,799 ± 2466	15,179 ± 2289	0.4956	5513 ± 1255	5936 ± 1065	0.8029	18,313 ± 3549	21,115 ± 2485	0.5340
Unfed
N	6	6		6	6		6	6
Body weight (g)							36.85 ± 2.12	37.01 ± 1.99	0.9586
Food intake (kcal)	0	0		0	0		0	0
Vo2 (ml/kg lean mass/h)	5643 ± 221.60	5644 ± 101.86	0.9970	4585 ± 230.51	4547 ± 157.97	0.8953	5115 ± 212.53	5091 ± 126.55	0.9234
Vco2 (ml/kg lean mass/h)	4027 ± 144.58	3996 ± 70.73	0.8508	3286 ± 154.03	3245 ± 106.04	0.8312	3657 ± 140.15	3617 ± 85.75	0.8125
RER (Vco2/Vo2)	0.7142 ± 0.0033	0.7080 ± 0.0019	0.1412	0.7174 ± 0.0033	0.7139 ± 0.0023	0.4137	0.7155 ± 0.0029	0.7107 ± 0.0018	0.1953
EE (kcal/kg lean mass/h)	26.49 ± 1.0231	26.46 ± 0.4750	0.9761	21.54 ± 1.0690	21.35 ± 0.7329	0.8838	24.02 ± 0.9832	23.88 ± 0.5880	0.9035
Total activity (beam breaks)	35,897 ± 6045	40,484 ± 5337	0.5822	12,202 ± 2746	12,706 ± 2495	0.8947	48,099 ± 8473	53,190 ± 7425	0.6612
Ambulatory activity (counts)	19,678 ± 3376	22,210 ± 2924	0.5834	5290 ± 1297	5383 ± 1143	0.9583	24,968 ± 4426	27,593 ± 3670	0.6580
Refeed
N	6	6		6	6		6	6
Body weight (g)							37.79 ± 2.06	37.57 ± 1.98	0.9405
Food intake (kcal)	11.11 ± 0.135*	9.48 ± 0.561*	0.0423*	3.44 ± 0.386	2.90 ± 0.312	0.3070	14.55 ± 0.4454*	12.39 ± 0.5515*	0.0155*
Vo2 (ml/kg lean mass/h)	5914 ± 239.53	6071 ± 125.66	0.5772	5201 ± 254.85	5187 ± 174.10	0.9657	5552 ± 239.42	5624 ± 145.89	0.8049
Vco2 (ml/kg lean mass/h)	4506 ± 157.98	4526 ± 81.21	0.9122	3984 ± 165.08	3904 ± 109.04	0.6957	4241 ± 155.71	4211 ± 92.23	0.8723
RER (Vco2/Vo2)	0.7629 ± 0.0054*	0.7458 ± 0.0040*	0.0311*	0.7675 ± 0.0074	0.7534 ± 0.0053	0.1536	0.7650 ± 0.0060	0.7493 ± 0.0044	0.0629
EE (kcal/kg lean mass/h)	28.11 ± 1.1074	28.74 ± 0.5772	0.6305	24.75 ± 1.1746	24.60 ± 0.7974	0.9179	26.41 ± 1.1045	26.64 ± 0.6687	0.8595
Total activity (beam breaks)	31,069 ± 8060	35,028 ± 5833	0.6999	12,508 ± 2680	11,956 ± 2066	0.8739	43,577 ± 10,661	46,984 ± 7839	0.8025
Ambulatory activity (counts)	15,373 ± 4644	17,250 ± 2722	0.7363	5174 ± 1418	4881 ± 852	0.8636	20,547 ± 6036	22,131 ± 3547	0.8267
HFD (male)
Ad libitum
N	6	6		6	6		6	6
Body weight (g)							44.71 ± 1.21	46.41 ± 2.74	0.5898
Food intake (kcal)	11.05 ± 0.485	10.92 ± 0.674	0.8820	3.69 ± 0.415	2.89 ± 0.326	0.1680	14.73 ± 0.5065	13.81 ± 0.7965	0.3621
Vo2 (ml/kg lean mass/h)	4896 ± 178.31	5268 ± 157.82	0.1494	4340 ± 179.53	4720 ± 163.00	0.1485	4615 ± 175.50	4992 ± 157.97	0.1426
Vco2 (ml/kg lean mass/h)	3803 ± 136.51	4071 ± 120.76	0.1729	3339 ± 131.94	3561 ± 122.98	0.2469	3569 ± 129.95	3814 ± 120.27	0.1971
RER (Vco2/Vo2)	0.7769 ± 0.0044	0.7728 ± 0.0031	0.4642	0.7697 ± 0.0034*	0.7545 ± 0.0041*	0.0181*	0.7735 ± 0.0031	0.7641 ± 0.0032	0.0600
EE (kcal/kg lean mass/h)	23.36 ± 0.8468	25.11 ± 0.7497	0.1532	20.67 ± 0.8468	22.39 ± 0.7719	0.1638	22.00 ± 0.8290	23.74 ± 0.7498	0.1516
Total activity (beam breaks)	19,968 ± 2233	18,985 ± 1879	0.7434	10,056 ± 446	9179 ± 1061	0.4721	30,024 ± 2392	28,164 ± 2693	0.6170
Ambulatory activity (counts)	8471 ± 1549	7870 ± 1280	0.7713	3449 ± 326	2999 ± 614	0.5364	11,920 ± 1768	10,869 ± 1810	0.6866
Unfed
N	6	5		6	5		6	5
Body weight (g)							41.54 ± 1.21	44.41 ± 2.82	0.3888
Food intake (kcal)	0	0		0	0		0	0
Vo2 (ml/kg lean mass/h)	4666 ± 216.33	4867 ± 194.46	0.5055	3693 ± 133.10	3930 ± 159.85	0.2869	4177 ± 170.75	4396 ± 162.82	0.3770
Vco2 (ml/kg lean mass/h)	3340 ± 147.61	3470 ± 137.69	0.5348	2659 ± 83.47	2806 ± 113.43	0.3308	2998 ± 112.11	3136 ± 113.67	0.4094
RER (Vco2/Vo2)	0.7162 ± 0.0027	0.7130 ± 0.0025	0.4004	0.7207 ± 0.0041	0.7139 ± 0.0021	0.1842	0.7182 ± 0.0033	0.7134 ± 0.0020	0.2482
EE (kcal/kg lean mass/h)	21.91 ± 1.0068	22.84 ± 0.9109	0.5107	17.37 ± 0.6102	18.45 ± 0.7493	0.2941	19.63 ± 0.7892	20.63 ± 0.7609	0.3825
Total activity (beam breaks)	17,821 ± 1731	22,326 ± 3195	0.2595	6245 ± 459	7229 ± 894	0.3654	24,066 ± 2046	29,555 ± 3826	0.2512
Ambulatory activity (counts)	7579 ± 890	10,425 ± 2360	0.3090	1672 ± 215	2318 ± 586	0.3466	9250 ± 1019	12,743 ± 2867	0.3028
Refeed
N	6	5		6	5		6	5
Body weight (g)							42.57 ± 1.15	45.43 ± 2.83	0.3890
Food intake (kcal)	11.14 ± 0.977	10.66 ± 0.898	0.7228	4.42 ± 0.318	3.28 ± 0.435	0.0690	15.56 ± 1.108	13.94 ± 0.8541	0.2759
Vo2 (ml/kg lean mass/h)	4992 ± 136.64	5113 ± 150.94	0.5682	4295 ± 129.02	4374 ± 122.97	0.6656	4639 ± 128.56	4738 ± 134.79	0.6089
Vco2 (ml/kg lean mass/h)	3764 ± 83.21	3849 ± 107.15	0.5454	3329 ± 75.07	3332 ± 89.30	0.9795	3544 ± 76.37	3587 ± 95.73	0.7344
RER (Vco2/Vo2)	0.7546 ± 0.0066	0.7531 ± 0.0031	0.8406	0.7761 ± 0.0079	0.7618 ± 0.0032	0.1406	0.7646 ± 0.0069	0.7572 ± 0.0027	0.3527
EE (kcal/kg lean mass/h)	23.68 ± 0.6204	24.25 ± 0.7067	0.5629	20.48 ± 0.5819	20.79 ± 0.5782	0.7166	22.07 ± 0.5817	22.50 ± 0.6314	0.6292
Total activity (beam breaks)	26,584 ± 5071	19,906 ± 1166	0.2506	8855 ± 1394	8000 ± 702	0.6000	35,440 ± 6372	27,905 ± 1726	0.2993
Ambulatory activity (counts)	12,476 ± 3090	8073 ± 846	0.2206	3173 ± 826	2498 ± 461	0.4965	15,649 ± 3888	10,571 ± 1263	0.2603

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At the time of the study, HFD-fed mice were 30 wk of age. HFD-fed mice were given 3 d to acclimate to the metabolic cages. Ad libitum data were collected on the fourth day. Mice were denied access to food but had free access to water on d 5 (unfed data), and refeed data were collected on d 6 when mice regained free access to food. *P < 0.05.

Impact of myonectin deletion on hematologic parameters

Myonectin (also referred to as erythroferrone) plays important roles in linking stress erythropoiesis and hepatic iron mobilization in response to blood loss or anemia by suppressing hepcidin expression in the liver (37, 38). Here, under basal nonstressed conditions, myonectin deficiency did not alter any blood parameters, including hemoglobin level, hematocrit, and population counts of lymphoid and myeloid cell types in male mice (Supplemental Table S3), consistent with previous findings (38). Interestingly, under basal conditions when mice were fed a control LFD, female mice lacking myonectin had significantly elevated total white blood cell counts as well as lymphocyte counts. Although diet-induced obesity can adversely affect multiple cell types in the immune system (69), metabolic stress from an HFD did not alter any of the blood parameters in myonectin-deficient mice of either sex relative to control WT littermates (Supplemental Table S3). Liver hepcidin expression was also indistinguishable between WT and KO mice (unpublished results). Thus, myonectin is not required for iron homeostasis under conditions in which stress erythropoiesis is not induced.

Myonectin deletion impairs lipid handling in diet-induced obese male mice

Although HFD-fed female WT and myonectin-KO mice had similar rates of lipid clearance following an oral lipid gavage (unpublished results), male KO mice exhibited significant impairment in uptake of TGs and free fatty acids from the circulation compared with WT littermates (Fig. 3). The impaired lipid clearance phenotype following lipid loading was confirmed in 2 additional independent cohorts of HFD-fed mice. Mice develop insulin resistance after exposure to an HFD for a short period (1–4 wk), whereas more pronounced differences in body weight gain occur after longer exposure to high-fat feeding (70). Because lipid tolerance was different between WT and KO male mice fed an HFD, we sought to determine whether insulin resistance due to HFD is sufficient to impair lipid disposal in response to oral lipid gavage. In contrast to chronic high-fat feeding, relatively short exposure to an HFD for 4 wk did not alter lipid tolerance in myonectin-deficient mice compared with WT littermates (unpublished results). This result suggests that excess adiposity from chronic high-fat feeding is necessary to alter lipid handling capacity in myonectin-null animals.

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Figure 3

Impaired lipid tolerance in myonectin-KO male mice fed an HFD. Serum TG (A) and NEFA (B) during lipid tolerance test in male mice (WT, n = 12; KO, n = 16) at 17 wk of age. *P < 0.05, **P < 0.01, ***P < 0.001.

Impact of myonectin deficiency on metabolic response to high-fat feeding later in life

Diet-induced obese mice in our studies were exposed to HFD beginning at 5 wk of age, which corresponds to adolescence. To determine whether myonectin is required for metabolic flexibility in adult animals, we exposed another cohort of mice to HFD after they reached 21 wk of age. After switching from standard chow to HFD for 12 wk, mice were assessed by several tolerance tests. Body weight and glucose and insulin tolerance did not differ between genotypes of either sex (Supplemental Fig. S2A–G). No differences in lipid tolerance were noted between WT and KO female mice (Supplemental Fig. S2H, I). Male myonectin-KO mice, however, exhibited reduced capacity to clear serum TG from the circulation following an oral lipid gavage (Supplemental Fig. S2J, K). The magnitude of difference between genotypes in lipid tolerance was smaller in this older cohort of myonectin-deficient male mice as compared with younger cohorts (Fig. 3). These results indicate that the severity of the lipid tolerance phenotype is contingent upon the age at which mice are exposed to the metabolic stress of high-fat feeding.

Elevated postprandial VLDL–TG levels in myonectin-deficient male mice

Although myonectin deficiency impairs the capacity to acutely handle orally delivered emulsified intralipid, it is unclear whether myonectin is required for the proper handling of dietary lipids in a postprandial state following physiologically normal ad libitum feeding. To test this, HFD-fed male mice were unfed overnight and then given free access to food for 2 h. Blood samples were taken before and after refeeding, and serum lipid levels were measured. Although fasting serum TG levels were similar between WT and KO mice, postprandial serum TG levels were significantly higher in myonectin-KO mice upon refeeding following an unfed period (Fig. 4A). Fasting and postprandial serum NEFA and cholesterol levels were not different between genotypes (Fig. 4B, C). Food intake did not differ between WT and KO mice (Table 4); thus, differences in calorie intake cannot explain differences in postprandial serum TG levels. Fractionation of pooled postprandial serum revealed that myonectin-KO male mice had significantly elevated and larger very LDL (VLDL)–TG particle levels compared with WT males (Fig. 4D). Lipoprotein cholesterol profiles showed that the size, but not the amount, of HDL cholesterol was larger in the myonectin-KO animals (Fig. 4E). These data indicate that myonectin is required for proper postprandial handling of dietary TG derived from normal ad libitum feeding of an HFD.

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Figure 4

Higher postprandial VLDL-TG levels in myonectin-KO male mice fed an HFD. A–C) Serum TG (A), NEFA (B), and cholesterol (C) levels in male mice after an overnight 14-h unfed period ("fasted") or after an unfed period overnight followed by ad libitum refeeding for 2 h (refed). Mice (36 wk of age) were euthanized after refeeding for tissue collection. A total of 2 WT mice did not eat during the refeed period as assessed by no increase in serum TG levels over fasting levels and an empty stomach. Therefore, refed data from these mice were not included in the analysis. Unfed: WT, n = 12; KO, n = 17. Refed: WT, n = 10; KO, n = 17. D, E) Refed serum samples from 10 mice of each genotype were pooled and subjected to FPLC fractionation to separate lipoprotein species. TG (D) and cholesterol (E) levels were measured in each fraction. IDL, intermediate-density lipoprotein. *P < 0.05.

Loss of myonectin does not affect intestinal lipid absorption and secretion

Several mechanisms could affect lipid clearance from the circulation. For example, elevated serum TG levels in myonectin-KO mice after oral lipid gavage or ad libitum feeding could be due to enhanced intestinal lipid absorption, greater secretion of TG-rich chylomicrons by the enterocytes, or both. To test this, overnight-unfed mice were injected with poloxamer 407, a detergent that inhibits LPL activity, preventing lipid uptake in peripheral tissue. After 1 h, mice were orally gavaged with intralipid. Accumulation of serum TG over time reflects intestinal lipid absorption and subsequent secretion of TG-rich chylomicrons. No differences in the kinetics of serum TG rise were noted between HFD-fed WT and KO mice (Supplemental Fig. S3A). Alternatively, impaired lipid uptake into peripheral tissues could result in the elevated TG levels seen in KO animals. LPL plays a major role in the cellular uptake of lipids derived from circulating lipoprotein particles; the enzyme is located on the surface of endothelial cells in most tissues and hydrolyzes TG to liberate free fatty acids, which can then be taken up by peripheral tissues (71). We therefore measured whole-body LPL activity in postheparin sera collected from mice in the postprandial state. As we previously observed, a 2-h ad libitum feeding following an overnight unfed period was sufficient to induce a difference in serum TG between genotypes; the heparin-displaceable total LPL activity, however, was not different between WT and myonectin-KO mice (Supplemental Fig. S3B, C).

Myonectin deletion alters adipose fat storage and lipid accumulation in the liver

We next sought to determine whether myonectin deficiency alters lipid storage in peripheral tissues. Although HFD-fed WT and KO mice had similar body weights and body compositions (Fig. 2A, B), the distribution of fat among tissues was altered. We noted that the KO mice (39 wk old) had significantly increased fat mass (both visceral and subcutaneous fat depots) and a modest reduction in liver weight (Fig. 5A–C and Supplemental Table S1). Liver histology revealed a marked reduction in steatosis in KO mice relative to WT littermates (Fig. 5D). Quantification of lipids also revealed significantly reduced hepatic TG content and lower cholesterol levels (Fig. 5E, F). Consistent with the histologic data and hepatic TG content, the lipid droplet–associated protein perilipin 2 was also correspondingly reduced in myonectin-KO livers (Fig. 5G). Histologic analysis of visceral epididymal WAT (eWAT) and subcutaneous iWAT indicated significantly larger adipocytes in myonectin-KO animals (Fig. 5H, I); this was further confirmed by cell size quantification (Fig. 5J, K). Differences in adipocyte cell size were more pronounced in the visceral fat depot (eWAT). These data suggest that myonectin plays a role in lipid partitioning between tissues and its deficiency promotes adipose tissue lipid storage and reduces lipid accumulation in the liver.

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Figure 5

Myonectin deficiency alters lipid distribution in male mice fed an HFD. A–D) WT and myonectin-KO mice were euthanized at 39 wk of age after a 2–4-h unfed period in the morning. Unless otherwise specified, data in this figure are from the analyses of WT (n = 12) and KO (n = 16) male mice. The wet weight of liver (A), visceral (gonadal) fat pad (B), and subcutaneous (inguinal) fat pad (C) are presented along with representative images of H&E-stained liver sections from WT and KO mice (D). E, F) Quantification of TG (E) and cholesterol (F) levels in livers. G) Western blot analysis of Perilipin 2. A total of 2 separate gels were run, each with WT (n = 6) and KO (n = 8) samples. Perilipin 2 (PLIN2) levels were first normalized to HSC70 levels from the same gel and then normalized to the mean WT value. Data were then combined from both gels for analysis. H, I) Representative images of H&E-stained eWAT (H) and iWAT (I) sections from WT and KO male mice. J) Quantification of adipocyte size in eWAT sections (WT, n = 5; KO, n = 5). K) Quantification of adipocyte size in iWAT sections (WT, n = 4; KO, n = 5). *P < 0.05, **P < 0.01.

Myonectin deletion alters multiple liver metabolite levels

Given the striking differences in lipid partitioning between adipose and liver of HFD-fed WT and KO animals, we measured circulating lipids from blood collected at the same time as tissue samples as shown in Fig. 5. Serum TG, NEFA, and β-hydroxybutyrate levels were similar between HFD-fed WT and KO male mice; serum cholesterol levels, however, were lower in myonectin-KO animals (Fig. 6A–D). To explore the genotypic difference in hepatic fat accumulation, we also performed unbiased metabolomics analysis to assess global changes in liver metabolites. Of the 642 metabolites quantified, only a small subset (32 in total) of metabolites significantly differed between WT and KO mice (Fig. 6E). Our data indicate that HFD-fed myonectin-KO male mice had elevated secondary bile acids, as well as increased levels of metabolites involved in 1-carbon metabolism. Interestingly, the hepatic level of imidazole propionate, a molecule reported to induce insulin resistance (72), was also significantly elevated in myonectin-KO male mice; however, this did not impair insulin sensitivity in male mice as measured by GTTs and ITTs (Fig. 2D, G). In addition to metabolite profiling, we performed extensive quantitative PCR to assess the expression of genes involved in lipid synthesis and catabolism in the liver and adipose tissue. Of the major genes involved in lipid synthesis and oxidation and lipoprotein and iron metabolism, there were no significant differences in the expression levels between genotypes in the liver or 2 major fat depots (Supplemental Fig. S4). However, for genes involved in bile acid metabolism, the expression of cholesterol 7 α-hydroxylase, the rate-limiting enzyme in bile acid synthesis, was significantly up-regulated in the liver of myonectin-KO male mice (Supplemental Fig. S4E).

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Figure 6

Steady-state serum and liver metabolite levels in WT and myonectin-KO male mice fed an HFD. Sera and livers were harvested from mice (39 wk old) 2–4 h after food removal. A–D) Quantification of serum TG (A), NEFA (B), cholesterol (C), and ketones (β-hydroxybutyrate) (D) (WT, n = 12; KO, n = 16). E) All liver metabolites that were significantly different (P < 0.05) between WT (n = 10) and myonectin-KO (n = 10) male mice. *P < 0.05.

Myonectin deficiency does not alter hepatic VLDL-TG secretion or fat oxidation

Because steady-state levels of metabolites (after 2 h of food removal) and gene expression could not fully explain the differential lipid accumulation in adipose and liver, we tested specific pathways of lipid flux in vivo. Reduced hepatic steatosis in HFD-fed KO mice could be due to increased hepatic VLDL-TG secretion. To test this, mice were first unfed for 4 h, beginning at 2 h into the light cycle, to ensure the absence of food-derived circulating chylomicrons secreted by enterocytes; in this physiologic state, most of the circulating TG is likely derived from VLDL-TG particles secreted from liver. Mice were next injected with poloxamer 407 to inhibit lipid uptake in peripheral tissue. Thus, the accumulation of serum TG over time reflects hepatic VLDL-TG secretion. This experiment revealed no differences between WT and KO mice, indicating that hepatic VLDL-TG secretion cannot account for the reduced hepatic TG accumulation in myonectin-deficient male mice (Supplemental Fig. S5A). Alternatively, enhanced hepatic fat oxidation can result in lower hepatic TG content. Although not a direct measure of fat oxidation in the liver, serum ketones are good proxies because ketogenesis in the liver is biochemically linked to fat oxidation (73); hence, circulating ketone levels correspond to the extent of hepatic fat oxidation. Circulating ketones (β-hydroxybutyrate) did not differ between WT and KO mice in either the unfed or fed state, indicating that there were no differences in hepatic fat oxidation between genotypes (Supplemental Fig. S5B).

Myonectin deficiency does not alter adipose tissue lipolysis

Given that myonectin-KO mice had greater adiposity, decreased adipose tissue lipolysis could account for this phenotype. Although recombinant myonectin treatment does not affect ex vivo lipolysis in WAT explants (28), the impact of its deficiency on adipose lipolysis in vivo is unknown. To test this, mice were injected with a β₃-adrenergic receptor agonist (CL 316,243) to stimulate lipolysis following a 5-h unfed period. CL-stimulated lipolysis was assessed by measuring serum glucose, glycerol, and NEFA before injection and 15 min postinjection; no differences were observed between WT and KO mice (Supplemental Fig. S6). Our data thus indicate that both basal (no changes in steady-state serum NEFA levels; Fig. 6B) and stimulated lipolysis were not altered in mice lacking myonectin.

The authors thank Susan Aja (Johns Hopkins University) for help with indirect calorimetry. This work was supported in part by a Grant from the U.S. National Institutes of Health (NIH), National Institute of Diabtes and Digestive and Kidney Diseases (DK084171 to G.W.W.). H.C.L. was supported by a National Research Service Award pre-doctoral fellowship (F31DK116537) and a NIH, National Institute of General Medical Sciences training grant (T32 GM007445). S.R. was supported by a postdoctoral fellowship from the American Diabetes Association (1-18-PMF-022). The authors declare no conflicts of interest.