Table 1.

In vitro/in vivo experiments and clinical studies on the association between GLP-1 analogs and C-cell pathology.

Authors [reference]	Studied drugs	Research materials or study subjects	Main outcomes investigated	Main findings
			Cellular models
Crespel et al. [32]	Glucagon, GLP-1 (7–36), and exendin (9–39)	Rat CA-77 C-cell line	cAMP production and calcitonin secretion	GLP-1 (7–36) and glucagon dose dependently stimulated cAMP production and calcitonin secretion. Exendin (9–39) abolished a further increase in cAMP formation at glucagon concentration over 10 nM and partially suppressed glucagon-induced calcitonin secretion.
Lamari et al. [31]	GLP-1 (7–37), and exendin (9–39)	Rat CA-77 C-cell line	cAMP production, calcitonin mRNA levels, and calcitonin secretion	GLP-1 (7–37) increased cAMP formation in a dose-dependent manner. Exedin (9–39), an antagonist of GLP-1 receptor, blunted the stimulation of cAMP production induced by GLP-1 (7–37). Gene expression and peptide secretion of calcitonin were increased after incubation of CA-77 cells with GLP-1 (7–37).
Knudsen et al. [11]	Liraglutide, exenatide, and GLP-1 (7–37)	Human TT C-cell line, rat MTC 6–23 C-cell line, and rat CA-77 C-cell line	GLP-1 receptor mRNA and protein expression; calcitonin release after GLP-1 receptor agonists	Native GLP-1, liraglutide, and exenatide all stimulate calcitonin gene expression and calcitonin secretion via the GLP-1 receptor in a dose-dependent manner in rat C cells. The human TT cells express few GLP-1 receptors compared with rat MTC 6–23 and CA-77 and show a lack of functional response to GLP-1 and GLP-1 receptor agonists.
			Animal experiments
(I) Rodents
Knudsen et al. [11]	Liraglutide versus vehicle control	Sprague Dawley rats aged 6-7 weeks and CD-1 mice at the age of 5–10 weeks	Plasma calcitonin and pathological examination to thyroid gland sections after dosing with liraglutide	Calcitonin levels increase with time and dose with 104-week repeated dosing of liraglutide. The incidences of both C-cell hyperplasia and C-cell tumor formation at 104 weeks were increased in a dose-dependent manner and reached statistical significance.
Madsen et al. [33]	Liraglutide, exenatide, and vehicle control	CD-1 wild-type mice aged 5-6 weeks and GLP-1-receptor knockout mice at the age of 4-5 weeks	Plasma calcitonin, pathological examination to thyroid tissue sections, and immunohistochemical staining for phosphoproteins after 13-week treatment with liraglutide or exenatide	GLP-1 agonists cause calcitonin release and C-cell hyperplasia in wild-type mice via a GLP-1-receptor-dependent mechanism. GLP-1 activates the mammalian target of rapamycin (mTOR) pathway by stimulating the production of cAMP. Activation of mTOR in turn results in downstream phosphorylation of ribosome S6. Liraglutide-induced C-cell hyperplasia in mice is not associated with RET activation.
(II) Primates
Knudsen et al. [11]	Liraglutide versus vehicle control	Cynomolgus monkeys aged 1-2 years	Plasma calcitonin and pathological analysis to thyroid gland sections after dosing with liraglutide	No increase in plasma calcitonin was seen in cynomolgus monkeys receiving a single dose of liraglutide or during 87-week daily dose. There was also no change in the thyroid gland sections, relative C-cell fraction of the thyroid gland, and proliferative index in the C cells after liraglutide for 52 weeks.
			Human studies
Gier et al. [10]	—	Human thyroid glands	Expression of GLP-1 receptors in tissue samples with C-cell abnormalities, papillary thyroid cancer, and normal thyroid	GLP-1 receptor immunoreactivities were detected in 33%, >90%, and 18% of patients with normal C cells, C-cell pathologies, and PTC lesions, respectively.
Hegedüs et al. [34]	Liraglutide versus active comparators and placebo	Nine clinical trials of 20–104-week duration	Geometric mean levels of serum calcitonin and outlier analysis	There was no significant difference in mean calcitonin levels between liraglutide and control groups. The proportions of subjects with calcitonin levels shifting to a higher category or above a clinically relevant cut-off value of 20 pg/mL were low and did not differ between treatment groups.
Elashoff et al. [36]	Exenatide versus rosiglitazone	Adverse effect reporting system	Overall thyroid cancer	Odds ratio for thyroid cancer was 4.73; P = 4 × 10−3