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. 2019 Feb:140:21-32.
doi: 10.1016/j.phrs.2018.11.013. Epub 2018 Nov 10.

Prenatal metformin exposure or organic cation transporter 3 knock-out curbs social interaction preference in male mice

Valentina R Garbarino  1 , Taylor A Santos  2 , Anastassia R Nelson  3 , Wynne Q Zhang  4 , Corey M Smolik  5 , Martin A Javors  6 , Lynette C Daws  7 , Georgianna G Gould  8
Affiliations

Prenatal metformin exposure or organic cation transporter 3 knock-out curbs social interaction preference in male mice

Valentina R Garbarino et al. Pharmacol Res. 2019 Feb.

Abstract

Poorly managed gestational diabetes can lead to severe complications for mother and child including fetal overgrowth, neonatal hypoglycemia and increased autism risk. Use of metformin to control it is relatively new and promising. Yet safety concerns regarding gestational metformin use remain, as its long-term effects in offspring are unclear. In light of beneficial findings with metformin for adult mouse social behavior, we hypothesized gestational metformin treatment might also promote offspring sociability. To test this, metformin was administered to non-diabetic, lean C57BL/6 J female mice at mating, with treatment discontinued at birth or wean. Male offspring exposed to metformin through birth lost social interaction preference relative to controls by time in chambers, but not by sniffing measures. Further, prenatal metformin exposure appeared to enhance social novelty preference only in females. However due to unbalanced litters and lack of statistical power, firm establishment of any sex-dependency of metformin's effects on sociability was not possible. Since organic cation transporter 3 (OCT3) transports metformin and is dense in placenta, social preferences of OCT3 knock-out males were measured. Relative to wild-type, OCT3 knock-outs had reduced interaction preference. Our data indicate gestational metformin exposure under non-diabetic conditions, or lack of OCT3, can impair social behavior in male C57BL6/J mice. Since OCT3 transports serotonin and tryptophan, impaired placental OCT3 function is one common mechanism that could persistently impact central serotonin systems and social behavior. Yet no gross alterations in serotonergic function were evident by measure of serotonin transporter density in OCT3, or serotonin turnover in metformin-exposed offspring brains. Mechanisms underlying the behavioral outcomes, and if with gestational diabetes the same would occur, remain unclear. Metformin's impacts on placental transporters and serotonin metabolism or AMPK activity in fetal brain need further investigation to clarify benefits and risks to offspring sociability from use of metformin to treat gestational diabetes.

Keywords: 1,1-dimethylbiguanide; Autism; Pregnancy; SLC22A3; Serotonin; Social behavior.

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Conflict of interest statement

Conflict of Interest Statement:

None of the authors have any conflicts of interest to report.

Figures

Fig. 1.
Fig. 1.. Glucose tolerance of pregnant dams is slightly impaired by metformin treatment.
A. Glucose tolerance test results from mid-gestation (ED 12-16) dams after 16 h (overnight) fast at baseline and 15 −120 min after injection of glucose (1 g/kg) did not reveal any differences between control (black circles, N = 5) or metformin (open boxes, N = 7) treatments at any specific time points. B. Comparison of area under curve (AUC) values from the glucose tolerance test revealed a slight but significant increase in metformin-treated dams relative to vehicle controls (* p < 0.05). Data are presented as means ± SEM.
Fig. 2.
Fig. 2.. Adolescent offspring weights at time of behavior tests.
Dam treatments were metformin in drinking water (0.5 mg/ml) stopped at birth (Met → birth), at wean (Met → wean) or control. Offspring weights (6-7 wk old) did not differ significantly. Boxes show 25th to 75th percentiles, center line is median, plus signs are means, whiskers are minimum to maximum weight. Weights for individuals are small points, all mice were used in the behavior tests, group size appears under boxes.
Fig. 3.
Fig. 3.. Gestational metformin exposure reduced male preference for social interaction.
There were no significant differences in female offspring sociability preference between groups. Within groups, a) time spent in stranger chambers (filled circles) vs. novel objects (open circles) was greater in female offspring of control dams (mean ± sem = 283 ± 17 vs. 212 ± 11 sec,*p < 0.05, N = 10) and dams treated with metformin through wean (Met-wean = 271 ± 15 vs. 170 ± 16 sec,**p<0.01, N = 10), but not from dams given metformin through birth (Met-birth = 279 ± 15 vs. 219 ± 18 sec, N = 8). b) Time spent sniffing stranger mice vs. novel objects was also greater in female offspring of control dams (119 ± 11 vs. 70 ± 5 sec,* p <0.05) and Met-wean (100 ± 10 vs. 51 ± 9 sec, *p < 0.05), but not Met-birth (146 ± 16 vs. 100 ± 14) exposed females. Male offspring from Met-birth dams spent significantly more time in novel object chambers and less time with strangers (# p < 0.05 in c) and exhibited a loss of social interaction preference in chamber entries (* p <0.05), but not in sniffing (d). Within groups c) time spent in stranger chambers (filled squares) vs. novel objects (open squares) was greater in male offspring of controls (mean ± sem = 301 ± 10 vs. 209 ± 10 sec,**p < 0.01, N = 16) but not in male offspring from Met - birth (231 ± 17 vs. 268 ± 11 sec, N = 8), or Met- wean (250 ± 28 vs. 231 ± 25 sec, N = 8) dams. d) In sniffing male offspring of controls displayed a preference for strangers vs. objects (130 ± 11 vs. 68 ± 8 sec, ** p <0.01) male offspring from Met-birth (123 ± 15 vs. 107 ± 15 sec) or Met-wean (118 ± 13 vs. 78 ± 13 sec) dams did not. Data are presented as symbols for novel object and stranger dwelling or sniffing times with a connecting line between measures of each individual animal.
Fig. 4.
Fig. 4.. Gestational metformin exposures had mixed effects on social novelty preference.
Considering all offspring, metformin exposure through birth enhanced or maintained within group social novelty preference, whereas exposure to metformin until wean impaired it. a) No within-group preference for new (dot in center circle) vs. old (filled circle) stranger chambers was evident in female offspring from control dams (mean ± sem = 230 ± 19 vs. 214 ± 24 sec, N = 10) or metformin stop at wean (Met-wean 252 ± 19 vs. 215 ± 18 sec, N = 10), whereas the metformin stopped at birth (Met-birth) group (325 ± 33 vs. 187 ± 20 sec, *p < 0.05, N = 8) preferred new strangers. b) Female offspring from Met-birth dams exhibited a significant preference for new strangers in sniffing time (159 ± 24 vs. 75 ± 10 sec, #(between) and *(within groups) p <0.05), whereas control (88 ± 11 vs. 70 ± 12 sec) or Met-wean female offspring (98 ± 11 vs. 64 ±10 sec) did not. c) Control and Met-birth male offspring spent more time in chambers with new (dot in center squares) vs. old (filled squares) strangers (control 303 ± 11 vs. 199 ± 13; N= 16, Met-birth 312 ± 12 vs. 162 ± 11 N = 8; *p < 0.05), but Met-wean male offspring (242 ± 32 vs. 234 ± 23, N = 8) did not. d). However based on sniffing, there was no preference for novelty exhibited by male offspring from control (115 ± 14 vs. 87 ± 9), met-birth (122 ± 11 vs. 65 ± 7) or met-wean (101 ± 19 vs. 77 ± 12) dams. Data are presented as symbols showing individual values with a connecting line between new and old stranger times for each individual mouse.
Fig. 5.
Fig. 5.. Organic cation 3 knock-out mice do not exhibit social interaction preference.
Male wild-type (WT, N = 16) and OCT3 knock-out (KO, N = 16) mouse social preferences were compared, and a) Social interaction preference by measure of time in chambers was significantly lower in OCT3 KO mice # p <0.05). Only WT spent more time in chambers with strangers (filled square) vs. novel objects (open square) WT mean ± sem = 301 ± 16 vs. 185 ± 16 sec, KO 257 ± 14 vs. 256 ± 13 sec (**p <0.01). b) By measure of sniff OCT3 KO social interaction preference was also significantly less than WT (# p <0.05), WT mean ± sem = 166 ± 14 vs. 86 ± 13 sec, KO 122 ± 10 vs. 89 ± 11 sec (**p <0.01; *p<0.05). c) No significant differences were observed between OCT3 genotypes for social novelty preference wherein time in chambers with new (square with dot in center) vs. old (filled square) strangers differences were compared. WT mean ± sem = 252 ± 26 vs. 228 ± 22 sec, KO 287 ± 18 vs. 211 ± 19 sec. d)By measure of time sniffing WT and KO did not differ in novelty preference, WT mean ± sem = 103 ± 8 vs. 84 ± 12, KO 113 ± 11 vs. 74 ± 8, but within KO group more time was spent sniffing new vs. old strangers (*p<0.05). Data are presented as symbols showing individual values with a connecting line between them for each individual mouse.
Fig. 6.
Fig. 6.. Males exposed to metformin through wean dropped more fecal boli during sociability testing.
Boli in the center chamber of the three chamber arena were tallied after social preference tests ended, and males in the metformin through wean (Met-wean) group dropped significantly more than control males (*p < 0.05). Boxes show 25th to 75th percentiles, center line is median, plus signs are means, whiskers are minimum to maximum fecal boli, # boli per individual are small symbols, sample sizes are shown as numbers under each box.
Fig. 7.
Fig. 7.. Chamber entries by metformin-exposed offspring during sociability tests.
(a) Female mice exposed to metformin through wean made significantly fewer entries during social interaction tests than controls (* p <0.05). (a) Male mice made comparable chamber entries across treatment groups in both test phases. Boxes show 25th to 75th percentiles, center lines are medians, plus signs are means, whiskers are minimum to maximum entries, entries of individuals are small symbols, sample sizes are shown as numbers under each box.
Fig. 8.
Fig. 8.. Marble burying behavior was unchanged by gestational metformin exposure.
Neither females nor males exhibited any difference in the number of marbles buried in bedding over 30 min. Boxes show 25th to 75th percentiles, center lines are medians, plus signs are means, whiskers are minimum to maximum buried, and number buried per individual are small symbols, sample sizes are shown as numbers under each box.
Fig. 9.
Fig. 9.. Serotonin transporter density in wild-type and OCT3 knock-out mouse terminal fields.
No significant differences were observed between genotypes in the brain regions measured. CPu = caudate putamen (striatum), Nu Acb = nucleus accumbens core, Hipp = hippocampus, DG = dentate gyrus, CA1 = CA1 region, CA3 = CA3 region. Boxes show 25th to 75th percentiles, center lines are medians, plus signs are means, whiskers are minimum to maximum densities, and individual density measures are small symbols , N = 6 per genotype.
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