Plasma concentrations of glucagon and glucagon-like peptide-1 are reduced in dogs with aminoaciduric canine hypoaminoacidemic hepatopathy syndrome versus healthy dogs: a preliminary study

Sarah M. Holm Loftus Laboratory, Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY

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Seth A. Peng Loftus Laboratory, Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY

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Marlena M. Holter Cummings Laboratory, Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY

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Bethany P. Cummings Cummings Laboratory, Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY
Center for Alimentary and Metabolic Science, Department of Surgery, School of Medicine, University of California, Sacramento, CA

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John P. Loftus Loftus Laboratory, Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY

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OBJECTIVE

To compare plasma concentrations of glucagon and glucagon-like peptide-1 (GLP-1) between healthy dogs and dogs with aminoaciduric canine hypoaminoacidemic hepatopathy syndrome (ACHES) dogs.

ANIMALS

Privately owned healthy (n = 5) control (CON) and ACHES (8; including 3 with diabetes mellitus) dogs enrolled between October 2, 2019, and March 4, 2020.

PROCEDURES

This was a prospective case-control study. Fasting and 15-minute postprandial plasma glucagon total GLP-1 concentrations were measured with commercial immunoassays.

RESULTS

Dogs with ACHES had lower fasting (median, 0.5; mean difference, 3.8; 95% CI, 0.52 to 7.0 pmol/L; P = .021) and postprandial (median, 0.35; mean difference, 5.0; 95% CI, 1.8 to 8.3 pmol/L; P = .002) plasma glucagon concentrations than CON (fasting and postprandial medians, 3.5 and 4.6 pmol/L, respectively). ACHES dogs had significantly (median, 4.15; mean difference, 12.65; 95% CI, 2.0 to 16.3 pg/ml; P = .011) lower postprandial plasma GLP-1 concentrations than CON (median, 16.8 pg/ml). There was no significant difference between fasting GLP-1 levels between the 2 groups.

CLINICAL RELEVANCE

Lower postprandial plasma GLP-1 concentrations may contribute to the propensity of diabetes mellitus in ACHES. Lower glucagon concentrations may reflect an appropriate physiologic response to hypoaminoacidemia.

Abstract

OBJECTIVE

To compare plasma concentrations of glucagon and glucagon-like peptide-1 (GLP-1) between healthy dogs and dogs with aminoaciduric canine hypoaminoacidemic hepatopathy syndrome (ACHES) dogs.

ANIMALS

Privately owned healthy (n = 5) control (CON) and ACHES (8; including 3 with diabetes mellitus) dogs enrolled between October 2, 2019, and March 4, 2020.

PROCEDURES

This was a prospective case-control study. Fasting and 15-minute postprandial plasma glucagon total GLP-1 concentrations were measured with commercial immunoassays.

RESULTS

Dogs with ACHES had lower fasting (median, 0.5; mean difference, 3.8; 95% CI, 0.52 to 7.0 pmol/L; P = .021) and postprandial (median, 0.35; mean difference, 5.0; 95% CI, 1.8 to 8.3 pmol/L; P = .002) plasma glucagon concentrations than CON (fasting and postprandial medians, 3.5 and 4.6 pmol/L, respectively). ACHES dogs had significantly (median, 4.15; mean difference, 12.65; 95% CI, 2.0 to 16.3 pg/ml; P = .011) lower postprandial plasma GLP-1 concentrations than CON (median, 16.8 pg/ml). There was no significant difference between fasting GLP-1 levels between the 2 groups.

CLINICAL RELEVANCE

Lower postprandial plasma GLP-1 concentrations may contribute to the propensity of diabetes mellitus in ACHES. Lower glucagon concentrations may reflect an appropriate physiologic response to hypoaminoacidemia.

Aminoaciduric canine hypoaminoacidemic hepatopathy syndrome (ACHES) is a recently defined condition occurring in dogs with hepatocutaneous-associated hepatopathy, hypoaminoacidemia, and variable aminoacidurias. Urine amino acid losses were most consistently lysinuria, methioninuria, or both, while other aminoacidurias such as proline were more variable.1 This syndrome is the most common cause of the unique skin lesions of superficial necrolytic dermatitis (SND).1 ACHES combined with SND is synonymous with hepatocutaneous syndrome. The ACHES acronym is derived from the salient clinical features, and the acronym itself is evocative of the common presence of painful SND lesions. Hepatocutaneous-associated hepatopathy and hypoaminoacidemia are consistent features of the syndrome. Veterinarians first described cases of SND in 19862 as an ulcerative dermatosis associated with diabetes mellitus (DM) in 4 dogs. The association of dogs with SND to DM has since been further established,13 and in 1 study,1 11/41 dogs with ACHES had concurrent DM. In humans, SND is commonly caused by glucagonomas, which are glucagon-secreting tumors.46 In both species, SND is accompanied by hypoaminoacidemia.1,5,79 The pathophysiology of SND in glucagonoma patients, dogs, and humans alike is attributable to excessive glucagon production promoting amino acid dysregulation. However, the underlying pathophysiologic drivers of ACHES remain poorly defined.

In dogs, SND is rarely attributed to glucagonomas; however, the similarities between ACHES and glucagonoma syndrome in humans suggest glucagon dysregulation may also be involved in ACHES. Glucagon and glucagon-like peptide-1 (GLP-1) are vital metabolic regulators of glucose and amino acid homeostasis or glucose, respectively. However, much of our understanding of these hormones derives from human or mouse models, and comparatively little is known about dogs. Glucagon increases glucose production and prevents hypoglycemia, whereas GLP-1 promotes beta-cell secretion of insulin and promotes glucose utilization.1013 We speculate that dogs with ACHES will experience an exaggerated secretion of glucagon that is similarly seen in animals with DM, pancreatic alpha-cell hyperplasia, and glucagonomas.

In addition to blood glucose regulation, glucagon’s relationship with amino acids is being increasingly recognized. Most amino acids stimulate glucagon secretion to various degrees.14 Amino acids form a feedback loop with glucagon, where increased glucagon signals through the G protein-coupled glucagon receptor, driving increased cellular uptake and decreased circulating concentrations of amino acids.1517 Various tissues express the glucagon receptor, including the liver, small intestine, brain, pancreatic beta cells, and kidney. This wide tissue distribution reflects glucagon’s importance for regulating metabolic pathways in diverse cell types. In 1 study,18 proximal renal tubular cells expressed the glucagon receptor and glucagon-stimulated tubular glucose reabsorption. Other studies19 corroborate that glucagon affects proximal renal tubular function. Thus, although direct evidence is lacking, glucagon could conceivably modulate amino acid transport and tubular reabsorption. Increased urinary amino acid clearance in a patient with glucagonoma supports this assertion.20

Glucagon levels in dogs with ACHES have not been directly compared with healthy dogs. Perturbations in glucagon signaling could help explain both the diabetic and hypoaminoacidemia phenomena in ACHES. Proglucagon is the shared precursor of glucagon, GLP-1, and GLP-2.21 GLP-1 opposes glucagon action by inhibiting glucagon secretion.22 GLP-1 is also an incretin hormone, which potentiates glucose-stimulated insulin secretion from beta cells to maintain glucose homeostasis. Lower concentrations of GLP-1 in human DM patients likely contribute to disease pathophysiology.23 Importantly, GLP-1 receptor agonists are pharmaceutically available, which could have a theoretical benefit for diabetic dogs with insulin resistance, for example, dogs with diabetic ACHES. A preliminary study evaluating an FDA-approved commercially available GLP-1 analog in healthy dogs suggested that GLP-1 analogs may be efficacious in improving glucose regulation in diabetic canine patients.2426

We hypothesized that dogs with ACHES have similar or higher glucagon and lower GLP-1 fasting concentrations and postprandial responses than healthy dogs. Therefore, we sought to compare plasma concentrations of glucagon and GLP-1 between healthy and ACHES dogs.

Materials and Methods

Case selection criteria

One investigator (JPL) identified all enrollment cases from enrollment in ongoing studies investigating ACHES between October 2, 2019, and March 4, 2020. Cases were recruited from clinics across the United States, including the authors’ institution. All case dogs had physical exams, complete blood counts, chemistry profiles, and urinalysis conducted. Inclusion criteria for the ACHES group align with the diagnostic criteria, overlapping with hepatocutaneous syndrome, in previous reports.1,3,2729 Briefly, cutaneous lesions were definitively diagnosed as SND by histopathology or clinically diagnosed by characteristic appearance and distribution. Hepatocutaneous-associated hepatopathy was definitively diagnosed by histopathology or clinically diagnosed by characteristic ultrasound (nodular, “Swiss cheese” appearance) and compatible enzymopathy (predominantly alkaline phosphatase increases). A pattern of hypoaminoacidemia consistent with ACHES was necessary for diagnostic inclusion. Urine amino acid profiles, although sometimes diagnostically useful, are not necessary for a definitive diagnosis and were therefore not mandated for inclusion. Dogs with concurrent regulated DM were permitted due to the association of DM with ACHES. Exclusion criteria included the following: (1) diagnosis of glucagonoma, (2) failure to develop skin lesions in cases without a histologic diagnosis, or (3) skin or liver histopathology results were inconsistent with SND or hepatocutaneous-associated hepatopathy. Healthy dogs (CON) owned by staff or students from Cornell University were prospectively enrolled in the study based on an unremarkable history, physical examination findings, and clinical pathology (minimally complete blood count and biochemistry) results. The Institutional Animal Care and Use Committee of Cornell University approved animal use for this study. Owner-informed consent was obtained for the enrollment of all dogs.

Sample collection

Blood was collected into sodium heparin tubes (4-mL BD Vacutainer) from patients with needles and venipuncture sites at the phlebotomists’ discretion. Heparinized plasma was obtained from centrifuged (according to clinical site protocols, typically 500 X g or greater for 10 to 15 minutes) whole blood at room temperature within 5 to 10 minutes of collection and frozen immediately (−80 °C for dogs sampled at Cornell University, approximately −20 °C for other sites). Samples from external sites were shipped on dry ice, typically within 24 to 48 hours of sampling, and then stored at −80 °C until assays were conducted. These methods meet or exceed (eg, storage at −20 °C is considered sufficient for glucagon) previous reports regarding GLP-130 and glucagon31 stability. A fasting (12 hours) sample (for amino acid profile and hormone measurements) and a 15-minute postprandial sample (for hormone measurements) were obtained from each dog. Dogs were fed meals they were normally fed, similar to mixed meal studies in people,3234 and diabetic dogs received their standard dose of prescribed insulin immediately upon meal completion.

Hormone assays

All assays were conducted at Cornell University. Total glucagon and GLP-1 were simultaneously measured in the same samples. The assays were conducted in 1 batch approximately 12 to 17 months after collection. Plasma glucagon was measured in fasting and nonfasting plasma samples using a commercially available kit (10-1281-01; Mercodia) per the manufacturer’s instructions. This sandwich-based ELISA assay has a detection range of 2 to 180 pmol/L (7 to 627 pg/mL) and a limit of 1.5 pmol/L (5.23 pg/mL), and the assay for the canine samples has been validated by the manufacturer (technical data on manufacturer’s website).35 All reagents were brought to room temperature before use. Calibrators were reconstituted with purified (Milli-Q; MilliporeSigma) water. Neat plasma and calibrators (10 µL) were plated in duplicate on precoated strip wells. The optical density was read at 450 nm using a microplate spectrophotometer (BioTek Epoch).

We measured total GLP-1 with a commercial assay kit (V-PLEX GLP-1 Total Kit; Meso Scale Diagnostics), per the manufacturer’s instructions. This assay has been validated by the manufacturer (white paper available on the manufacturer’s website)36 to measure active and inactive GLP-1 in plasma across 5 species, including canine, with a sensitivity of 0.017 to 120 pg/mL. The plate was read with a proprietary instrument (MESO QuickPlex SQ 120) within 30 minutes.

Amino acid profiles

Fasting plasma amino acid profiles were performed by a veterinary reference laboratory (Amino Acid Laboratory, University of California-Davis) as previously described for ACHES dogs.1,27

Statistical analysis

A lack of adequate glucagon or GLP-1 data measured with these assays in this condition precluded a useful a priori power analysis. Proportions and percentages were used to describe categorical data. Due to the small sample size, normality testing was not conducted, and the median and range were used to describe continuous data. The Mann-Whitney test was used to compare the ages between ACHES and CON groups. We used the 2-way ANOVA with matching and the Sidak test (with adjusted P values) to compare fasting and postprandial hormone concentrations between control and ACHES groups. The fasting to postprandial change in hormone concentrations was compared between CON and ACHES groups with the Mann-Whitney test. A correlation matrix with the Spearman test was conducted to evaluate relationships between individual dog hormone results and plasma amino acid concentrations. Spearman r values were interpreted as the following relationship strengths: 0.00 to 0.19 = very weak, 0.20 to 0.39 = weak, 0.40 to 0.59 = moderate, 0.60 to 0.79 = strong, and 0.80 to 1.0 = very strong.37 We reported strong and very strong relationships with significant P values. Commercial software (Prism 9.0 or later; GraphPad) computed the statistical analyses and generated corresponding graphs. A P value < .05 established significance.

Results

Case demographics

Eight dogs with ACHES (including 3 with ACHES and DM) and 5 healthy controls (CON) were included. The CON dog ages ranged from 8 to 12 years (median, 10 years) and included small mixed breed dogs (n = 2), Shih Tzu (1), Springer Spaniel (1), and Boxer (1). Two ACHES dogs (including 1 with diabetes) were diagnosed and treated at the authors’ institution. The remaining cases were managed at various primary care and specialty referral practices. No dogs were excluded. The ages of ACHES dogs ranged from 8 to 15 (median 11.5) years. There was no significant difference in age between ACHES and CON groups. Breeds included a small mixed breed dog (n = 1), large mix-breed dogs (3), a Welsh Springer Spaniel (1), a West Highland White Terrier (1), a Maltese (1), and Toy Poodle (1). All ACHES dogs in this study had SND skin lesions at diagnosis. All 3 diabetic dogs were treated with insulin, and no dogs were reported to be receiving glucocorticoids.

Hormone concentrations

Dogs with ACHES had lower fasting (median, 0.5; mean difference, 3.8; 95% CI, 0.52 to 7.0 pmol/L; P = .021) and postprandial (median, 0.35; mean difference, 5.0; 95% CI, 1.8 to 8.3 pmol/L; P = .002) plasma glucagon concentrations compared to healthy controls (Figure 1). The absolute postprandial change in plasma glucagon concentrations was not statistically different between the 2 groups (CON median = 0.6 pmol/L; ACHES median = 0.05 pmol/L). Similarly, the percent change in fasting and postprandial plasma glucagon concentrations was not significantly different between groups (data not shown). Dogs with ACHES had significantly (median, 0.35; mean difference, 5.0; 95% CI, 1.8 to 8.3 pmol/L; P = .002) lower postprandial plasma GLP-1 concentrations than CON dogs (fasting and postprandial medians, 3.5 and 4.6 pmol/L, respectively). There was no significant difference between fasting GLP-1 levels between the 2 groups. While the median postprandial change in GLP-1 plasma concentrations in ACHES cases (1.3 pg/mL) was lower than CON, this difference did not achieve statistical significance.

Figure 1
Figure 1

Fasting and 15-minute postprandial plasma glucagon and glucagon-like peptide GLP-1 concentrations in privately owned healthy dogs or with aminoaciduric canine hypoaminoacidemic hepatopathy syndrome ACHES in a case-control study conducted between October 2, 2019, and March 4, 2020. A—Plasma glucagon concentrations in healthy control (CON; n = 5) and ACHES dogs (8). B—Change in plasma glucagon concentrations between 12-hour fasting (PRE) and 15 minutes postprandial (POST; dogs fed their typical meal) in CON and ACHES dogs. C—Plasma GLP-1 concentrations in CON and ACHES dogs. D—Change in plasma GLP-1 concentrations between PRE and POST in CON and ACHES dogs. Each circle represents results for 1 or more dog; brackets represent comparisons made between groups. A and C—Within each group, a line connects the PRE and POST results for each dog. B and C—The line in each individual-value plot represents the median change. The dotted line represents no change. ns = No significant difference. *P < .05, **P < .01.

Citation: American Journal of Veterinary Research 84, 4; 10.2460/ajvr.22.10.0174

Amino acid profiles

Theresults of fasting amino acid profiles were consistent with those previously reported for dogs with ACHES (Table 1). Fasting glucagon concentrations were significantly and strongly associated with concentrations of several amino acids (Table 2). Significant, strong correlations were found between postprandial glucagon and l-ornithine and taurine. In contrast to glucagon, amino acid concentrations were correlated with fasting GLP-1; however, several fasting amino acid concentrations were associated with postprandial GLP-1 levels. The change in total plasma GLP-1 was associated with fasting plasma concentrations of l-arginine, l-asparagine, l-ornithine, and l-proline.

Table 1

Summary of plasma amino acid profile results for 8 privately owned dogs with aminoaciduric canine hypoaminoacidemic hepatopathy syndrome (ACHES) in a case-control study conducted between October 2, 2019, and March 4, 2020.

Amino acid Median (nmol/mL) Minimum (nmol/mL) Maximum (nmol/mL) Reference values (nmol/mL)
l-Alanine 88.1 63 589 289 ± 9
l-Arginine 26.8 12 153 102 ± 2
l-a-Amino-n butryic acid 12.5 3 31.7 6 ± 2
l-Asparagine 18 12.2 61 41 ± 1
l-Aspartic acid 2.9 0.7 6 7 ± 0.2
l-Citrulline 8.35 2 117 41 ± 2
Cystathionine 2.75 1 6.5 3 ± 1
l-Cystine 1 0 2.3 46 ± 1
l-Glutamic acid 23.1 16 169 24 ± 1
l-Glutamine 146 70.5 695 495 ± 9
Glycine 62.05 41 192 266 ± 8
l-Histidine 55.35 31.4 107 71 ± 2
1-Methyl-l-histidine 5.15 2.8 18 NA
3-Methy-l-histidine 6 3.4 49 6 ± 1
l-Isoleucine 37.2 23 109 51 ± 1
l-Leucine 66.2 31 187 120 ± 3
l-Lysine 54.25 43 262 131 ± 5
l-Methionine 21.5 5.2 62 57 ± 2
l-Ornithine 3.55 0 34 35 ± 2

Values are means ± SE.

NA = not available.

Table 2

Relationships between selected plasma amino acid and hormone concentrations in 8 privately owned dogs with ACHES described in Table 1.

Fasting glucagon (pmol/L) Postprandial glucagon (pmol/L) Fasting total GLP-1 (pg/mL) Postprandial total GLP-1 (pg/mL) Change in total GLP-1
Amino acid P r P r P r P r P r
l-Arginine .34 0.39 .22 0.50 .39 0.36 .03* 0.79 .05 0.74
l-Asparagine .18 0.53 .05 0.72 .95 −0.04 .02* 0.79 .02 0.83
l-Aspartic acid .01* 0.85 .12 0.61 .30 0.42 .01* 0.86 .13 0.59
l-Glutamic acid .03* 0.81 .17 0.55 .11 0.62 .02* 0.83 .24 0.48
1-Methyl-l-histidine .04* 0.76 .54 0.25 .15 0.57 .15 0.57 .46 0.31
l-Leucine .05* 0.73 .35 0.38 .15 0.57 .06 0.71 .30 0.43
l-Lysine .53 0.27 .07 0.69 1.00 0.00 .03* 0.79 .01 0.88
l-Ornithine .01* 0.84 .04* 0.75 .27 0.45 .003* 0.92 .14 0.58
l-Phenylalanine .04* 0.75 .11 0.62 .61 0.22 .03* 0.81 .09 0.66
l-Proline .26 0.46 .16 0.56 .88 −0.07 .10 0.64 .04 0.76
l-Serine .03* 0.81 .24 0.47 .13 0.60 .05* 0.74 .36 0.38
Taurine .06 0.71 .05* 0.74 .66 0.19 .02* 0.83 .08 0.67
l-Threonine .005* 0.90 .21 0.51 .17 0.55 .04* 0.76 .30 0.43
Tryptophan .01* 0.88 .11 0.62 .24 0.48 .02* 0.83 .22 0.50
l-Tyrosine .03* 0.81 .33 0.40 .20 0.52 .05* 0.74 .22 0.50
l-Valine .05* 0.73 .35 0.38 .13 0.60 .05* 0.74 .27 0.45

Amino acids without a robust (P < .05 and r > 0.59) relationship to hormones were omitted for clarity. The change in glucagon was not robustly associated with any amino acids. Spearman r values were interpreted as the following relationship strengths: 0.00–0.19 = very weak, 0.20–0.39 = weak, 0.40–0.59 = moderate, 0.60–0.79 = strong, and 0.80–1.0 = very strong.

*

P < .05.

Discussion

We aimed to evaluate plasma concentrations of glucagon and GLP-1 in dogs with ACHES compared to healthy dogs. We found that dogs with ACHES had significantly lower fasting and postprandial glucagon and postprandial GLP-1 plasma concentrations than healthy controls. These results refuted our hypothesis that glucagon concentrations are similar or higher in dogs with ACHES than in healthy dogs. However, they corroborate our hypothesis that GLP-1 concentrations are lower in this syndrome.

Previous glucagon concentrations reported for dogs with ACHES or SND were normal to slightly decreased; however, canine-specific reference ranges have been lacking.9,38 Several years ago, these blood glucagon concentrations were reported in SND cases and employed different analytical methods. Newer assays, including the one used in our study, provide superior specificity for glucagon by minimizing cross-reactivity with other proglucagon-derived peptides. The glucagon assay kit utilized in this study is not canine specific; however, the manufacturer has validated the assay for canine samples (technical data available on the manufacturer’s website)35 and demonstrates superior handling of matrix effects, ie, interference in samples from unknown causes, and different sample types (C Jones, BA, President, Mercordia, email, July 7, 2022). While our data do not support the hypothesis that normal but inappropriate glucagon concentrations are linked to ACHES pathophysiology, it is intriguing to consider if glucagon receptor function could be abnormal in afflicted dogs and may warrant further investigation.

Positive associations between hormone and plasma amino acid concentrations in ACHES dogs align with known secretagogue functions. However, it is crucial to interpret these findings in the context of recognizing that fasting amino acids were measured. Arginine is a secretagogue of glucagon and GLP-1 and was strongly associated with GLP-1 postprandial concentrations and excursion.39,40 Fasting arginine concentrations were not associated with absolute glucagon concentrations or postprandial change, suggesting that basal plasma concentrations have little influence on glucagon secretion. Glutamate (glutamic acid) also stimulates glucagon secretion,41 which was associated with fasting glucagon concentrations. In addition to l-arginine, l-ornithine stimulates GLP-1 secretion, and fasting plasma levels of both were associated with higher postprandial GLP-1 concentrations.15,40,42 Nutritional targets for increasing GLP-1 secretion in people have been described,43 and this may help support the rationale for some nutritional supplementation strategies already implemented for ACHES3 and pave the way for others. However, associations between nutrients and hormone concentrations do not indicate a cause-effect relationship and could suggest that higher hormone levels support higher plasma concentrations of several amino acids. This is less likely the case as increasing glucagon concentrations typically reduce the plasma concentrations of most amino acids, and GLP-1, unlike GLP-2, is not known to contribute to plasma amino acid homeostasis.44,45 Beyond nutritional strategies to increase GLP-1 secretion, GLP-1 receptor agonists could have a theoretical benefit for ACHES cases and warrant future consideration.

The enteroendocrine cells of the gastrointestinal tract cosecrete GLP-2 with GLP-1 on an equal molar basis.46 Thus, plasma GLP-1 concentrations could provide a surrogate marker for GLP-2 concentrations under the assumption that postsecretion distribution and elimination occur at similar rates. GLP-2 signaling increases intestinal amino acid absorption in mouse models.47 To the author’s knowledge, the role of GLP-2 in amino acid transport in the renal tubules has not been elucidated. However, decreased plasma GLP-2 concentration in ACHES dogs could theoretically contribute to the pathophysiology of aminoaciduria and hypoamminoacidemia in these patients. Therefore, if lower GLP-1 concentrations in ACHES primarily reflect reduced secretion, lower levels of secreted GLP-2 are anticipated. Unfortunately, a sufficiently sensitive assay to measure GLP-2 in the dog is unavailable to currently explore this postulation.

One limitation of this study is its small sample size, which could result in underpowered or extreme data. However, we applied statistical methods appropriate for small sample sizes.48 Cases of ACHES also included those with and without DM; however, this reflects ACHES demographics.1 A more extensive study comparing these groups separately and to a cohort of dogs with DM but no evidence of ACHES would likely provide additional insight into relationships between plasma GLP-1 concentrations and these disease states. Diets in this study were not controlled, and variability in dietary constituents could affect the postprandial changes in plasma GLP-1 and glucagon concentrations. However, fasting hormone trends were also lower in ACHES patients suggesting dietary nutrient variability is unlikely to alter the overall interpretation of these results. All patients were fed their typical diets due to concern for altered glycemic control in diabetic patients and the use of mixed meals in human studies3234 investigating alpha- and beta-cell responses; however, future studies could employ standardized diets and nutritional supplements to elucidate dietary influences on glucagon and GLP-1 concentrations in dogs. The 15-minute postprandial timepoint was selected based on the experience of 1 of the authors (BPC) in other species. A time course in clinical cases would be logistically challenging but would be required to identify an optimal postprandial sample collection time point. Finally, the nature of this study introduced some variability in sample handling that could affect analyte stability. However, all samples were kept cool and frozen as soon as possible, and we measured total GLP-1, which is more stable and abundant in systemic circulation than active GLP-1.49 The long-term stability of GLP-1 is high over a 1-year period; in contrast, glucagon recovery measured by radioimmunoassay was reduced by approximately 50% after freezing but remained between 6 and 12 months of storage.30 However, other investigators have not encountered this phenomenon with glucagon measured by the Mercordia ELISA (R Coch, MD, Instructor in Clinical Medicine, Weill Cornell Medicine, email, December 1, 2022), and studies using the same assay have also been conducted on frozen samples.50,51 Additionally, sample handling was similar for control and ACHES cases, further supporting the value in our comparative analyses.

The reduced GLP-1 plasma concentrations in this cohort of dogs with ACHES warrant further exploration into its role in ACHES. Correlations between known amino acid secretagogues of glucagon and GLP-1 suggest that dietary manipulation of arginine, glutamate, or ornithine is a rational target for treatment.

Acknowledgments

The authors thank Drs. Nicolas Berryessa, Martha Cline, Danielle Davignon, Tara Ghormley, Nicole Guma, and Polina Vishkautsan for providing case samples.

This study was funded by a Research Grant in Animal Health from Cornell University.

The authors declare that there were no conflicts of interest.

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