DOI: 10.5061/dryad.stqjq2c33
title: Insulin infusion is linked to increased NPPC expression in muscle and plasma C-type natriuretic peptide levels in dogs

description: 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.
description: Animal Care and Surgical
 Procedures Four conscious
 dogs (<i>Canis lupus familiaris</i>) 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 &lt;
 18,000/mm<sup>3</sup>, hematocrit &gt; 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
 &lt;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 &lt; 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.