The purpose of this study was to assess insulin-stimulated gene expression in canine skeletal muscle with a particular focus on NPPC, the gene that encodes C-type natriuretic peptide, a key hormonal regulator of cardiometabolic function. Four conscious canines underwent hyperinsulinemic, euglycemic clamp studies. Skeletal muscle biopsy and arterial plasma samples were collected under basal and insulin-stimulated conditions. Bulk RNA sequencing of muscle tissue was performed to identify differentially expressed genes between these two steady-state conditions. Our results showed that NPPC was the most highly expressed gene in skeletal muscle in response to insulin infusion, rising fourfold between basal and insulin-stimulated conditions. In support of our RNA-sequencing data, we found that raising the plasma insulin concentration 15-fold above basal elicited a 2-fold (p = 0.0001) increase in arterial plasma concentrations of N-terminal prohormone C-type natriuretic peptide. Our data suggest insulin may play a role in stimulating secretion of C-type natriuretic peptide by skeletal muscle. In this context, C-type natriuretic peptide may act in a paracrine manner to facilitate muscle-vascular bed crosstalk and potentiate insulin-mediated vasodilation. This could serve to enhance insulin and glucose delivery, particularly in the postprandial absorptive state.

Animal Care and Surgical Procedures

Four conscious dogs (Canis lupus familiaris) of either sex were studied. Dogs were housed in a facility that met the standards of the American Association for the Accreditation of Laboratory Animal Care guidelines. They were fed a 65-75 kcal/kg/day diet of canned meat and chow (28% protein, 49% carbohydrate, and 23% fat) and the protocol was approved by the Vanderbilt University Medical Center Animal Care Committee. Approximately 16 days prior to experiments, a silastic sampling catheter was placed in the femoral artery and a laparotomy was performed to place infusion catheters in the jejunal and splenic veins, which drain into the hepatic portal circulation, as previously described(22). Experimental inclusion criteria included a leukocyte count < 18,000/mm³, hematocrit > 36%, good appetite, normal bowel movements, and healthy physical appearance.

Experimental Procedures

The dogs were fasted for 18-hours prior to each experiment. On the morning of study, intravenous angiocatheters were placed in the cephalic and saphenous veins for infusion of human insulin, 20% dextrose, and somatostatin. The distal ends of the intraportal catheters and flow probes were exteriorized from their subcutaneous pockets through incisions made under local anesthesia (2% lidocaine). Dogs rested in a Pavlov harness throughout the study.

Each experiment consisted of a 90-minute resting period, a 30-min basal sampling period, and two 150-minute infusion periods where insulin (Novo Nordisk A/S, Copenhagen, Denmark) and somatostatin (Bachem, Torrance, CA) were infused in a peripheral vein and glucagon (Eli Lilly, Indianapolis, IN) was infused into the portal vein (with rates as shown on the top of Figure 1A). Plasma hormone and metabolite samples were collected during the final 30 minutes of each infusion period. Insulin was infused to raise the peripheral circulation plasma insulin concentration 4-fold and 25-fold above basal during the first and second infusion periods, respectively. Intraportal glucagon was infused at a rate to maintain the hormone’s plasma concentration at a basal level throughout the study.

At two points, at the end of the basal period just prior to infusing hormones and immediately following collection of the final plasma hormone and metabolite sample, a <1 cm scalpel incision was made over the biceps femoris muscle and a small muscle biopsy was collected under local anesthesia using sterile technique. Muscle samples were immediately flash frozen in liquid nitrogen and stored at -80°C.

RNA Sequencing

After completing all canine studies, we dissected twelve frozen muscle samples weighing between 8-19 mg each. Total RNA was extracted and purified using Qiagen’s All prep kit. Sequencing ready libraries were prepared using Takara SMARTseq v4 for cDNA synthesis kit and addition of sequencing ready adaptors with Nextera XT prep kits respectively.  Libraries were sequenced on an Illumina NovaSeq 6000 to an approximate sequencing depth of 40 million total reads.

Read alignment was performed using STAR (v2.7.3a) aligner. The raw read counts were estimated using HTSeq (v0.11.2). Read counts were normalized using DESeq2 to get the normalized counts. Additionally, the aligned reads were used for estimating expression of the genes using cufflinks (v2.2.1). Distribution of mapped reads was performed using RSeQC and RNA-SeQC tools. Analysis was performed using R and additional packages which included: ggplot2, reshape2 and ggrepel, corrplot, gplots and heatmap.2 on normalized counts of all protein coding genes for each sample. Differential expression analysis was performed using DESeq2 (R Bioconductor package, version 3.11). The RNA sequencing data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus(23) and are accessible through GEO Series accession number GSE167026 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE167026).

Metabolite and Hormone Assays

Arterial plasma glucose levels were assayed using the glucose oxidase reaction (Analox Instruments, Stourbridge, UK). Arterial plasma insulin (Millipore Cat# PI-12K, RRID:AB_2801580, MilliporeSigma, Burlington, MA, USA) and glucagon (Millipore Cat# GL-32K, RRID:AB_2757819, MilliporeSigma) concentrations were measured by radioimmunoassay. Arterial plasma NT-proCNP concentrations were measured using a sandwich ELISA (Biomedica Cat# BI-20812, RRID:AB_2811290, Biomedica, Vienna, Austria).

Statistics

For selecting differentially expressed genes, we used a false-discovery rate adjusted p-value of < 0.05 and fold change cut offs of ± 2. A paired t-test was used to compare mean arterial plasma concentrations of NT-proCNP between the basal and second insulin-stimulated periods. Simple linear regression was used to analyze the correlation between arterial plasma insulin and NT-proCNP concentrations. Statistical analysis was computed using GraphPad Prism version 8.4.3. Data are summarized as means ± standard deviations unless otherwise indicated. Some or all data generated or analyzed during this study are included in this published article or in the data repositories listed in References.