2020 Volume 67 Issue 6 Pages 593-605
Thyroid hormone is crucial for regulating lipid and glucose metabolism, which plays essential role in maintaining the health of pregnant women and their offspring. However, the current literature is just focusing on the development of offspring born to the untreated mothers with hypothyroidism, rather than mothers themselves. Additionally, the interaction between hypothyroidism and pregnancy, and its impact on the women’s health are still elusive. Therefore, this study was designed to compare the metabolic differences in dams with hypothyroidism starting before pregnancy and after pregnancy. Pre-pregnant hypothyroidism was generated in 5-week-old female C57/BL/6J mice using iodine-deficient diet containing 0.15% propylthiouracil for 4 weeks, and the hypothyroidism was maintained until delivery. Gestational hypothyroidism was induced in dams after mating, using the same diet intervention until delivery. Compared with normal control, gestational hypothyroidism exhibited more prominent increase than pre-pregnant hypothyroidism in plasma total cholesterol and low-density lipoprotein cholesterol, and caused hepatic triglycerides accumulation. Similarly, more significant elevations of protein expressions of SREBP1c and p-ACL, while more dramatic inhibition of CPT1A and LDL-R levels were also observed in murine livers with gestational hypothyroidism than those with pre-pregnant hypothyroidism. Moreover, the murine hepatic levels of total cholesterol and gluconeogenesis were dramatically and equally enhanced in two hypothyroid groups, while plasma triglycerides and protein expressions of p-AKT, p-FoxO1 and APOC3 were reduced substantially in two hypothyroid groups. Taken together, our current study illuminated that gestational hypothyroidism may elicit more pronounced lipid dysregulation in dams than dose the pre-pregnant hypothyroidism.
HYPOTHYROIDISM is a metabolic disease which is characterized by low level of thyroid hormone (TH) and high level of thyroid-stimulating hormone in serum [1]. TH plays an important role in regulating lipid and glucose metabolism and insulin signaling [2-4]. According to previous literature, TH stimulates cholesterol and other lipid synthesis, increases mobilization of plasma cholesterol and triglycerides (TG) [5-7]. Recent researches indicated that hypothyroidism is closely associated with non-alcoholic fatty liver disease (NAFLD) independently, which may involve in altering intracellular TH action [8, 9]. Furthermore, thyroid dysfunction is commonly related to changes in plasma lipid levels [10]. Patients with hypothyroidism are frequently accompanied by hypercholesterolemia and a remarkable increase in low-density lipoproteins (LDL-C), which may result from the decrease in the number and activity of LDL receptors (LDL-R) in liver [11, 12]. Experimental hypothyroidism in rats treated with propylthiouracil (PTU) showed an increase in plasma LDL-C, and decrease in plasma TG [13], which can be normalized by substitution therapy with TH [14].
The prevalence of hypothyroidism in women at reproductive age is relatively high, which is connected with hyperprolactinemia, menstrual abnormalities and anovulation in some cases [15]. Hence, pregnancy has been regarded as infrequent in untreated women with hypothyroidism [16]. However, it has been reported that some hypothyroid women with insufficient or no treatment were still able to carry pregnancy to full terms [17, 18]. On the other hand, hypothyroidism is also the most common diseases driven by pregnancy, affecting 3–5% of all pregnant women [19]. It’s worth mentioning that both women with hypothyroidism before pregnancy and those diagnosed to have hypothyroidism during pregnancy have experienced the short and long-term adverse consequences for themselves and their offspring [20]. Untreated mothers with hypothyroidism might have the increased risks of anemia, miscarriage, premature delivery, placental abruption, gestation-induced hypertension and pre-eclampsia, gestational diabetes, postpartum hemorrhage and adverse offspring outcomes such as, low birth weight, stunted growth, and mental retardation [21-23]. Nevertheless, not all studies have found an increased risk of adverse outcomes for mothers with hypothyroidism or for their offspring [22]. Meanwhile, previous researches suggested that untreated pregnant women with hypothyroidism may develop hypothyroidism and diabetes in the years after pregnancy [24, 25]. Considerable research efforts have been devoted to untreated experimentally-induced maternal hypothyroidism that might have serious consequences for offspring development like behavior, skin, brain [26-28]. However, few systematic studies have concerned the interaction between hypothyroidism and gestation. Therefore, the objective of the current study was to compare the differences of hepatic protein expression, insulin signaling, lipid and glucose metabolism between the pre-pregnant hypothyroidism and gestational hypothyroidism in dams after delivery. Taken together, our current data demonstrated that gestational hypothyroidism may elicit more obvious lipid dysregulation in dams after delivery than the pre-pregnant hypothyroidism.
Animal procedures were approved by the Animal Care and Use Committee, faculty of science, Anhui Medical University, in accordance with the international guiding principles for biomedical research involving animals of CIOMS.
Male and female mice of the C57BL/6J were purchased at 4 weeks of age from Changzhou Cavens, China. All mice were raised in the specific-pathogen-free (SPF) laboratory under conditions of controlled temperature (23 ± 2°C) and relative humidity (50–60%) with a 12 h light/dark cycle. After one week of acclimatization, female mice (16.8 ± 0.8 g) were randomly divided into pre-pregnant hypothyroidism group (PH), gestational hypothyroidism group (GH) and normal control group (NC). The mice in PH group received iodine-deficient diet containing 0.15% propylthiouracil [29] (IDP diet, cat. #D18503, Jiangsu Xietong Organism Co, Jiangsu, China) and normal drinking water for 4 weeks before pregnancy, while standard irradiated diet (cat. #D10001, Jiangsu Xietong Organism Co, Jiangsu, China) and water were available ad libitum to the mice in GH and NC groups, simultaneously. The murine body weights and fasting plasma glucose (FPG) levels were measured each week. Serum samples were obtained (n = 6/group) after four weeks of treatment for detecting free triiodothyronine (FT3), free thyroxine (FT4) and thyroid-stimulating hormone (TSH). At age of 10 weeks, female mice were placed together with males at a ratio of 2:1. The presence of a vaginal mucous plug in next morning after mating was considered as an index of pregnancy and this day was defined as the start of gestational days (GD0). The mice in PH and NC groups during pregnancy continued to receive the diet and water identical to pre-pregnancy, but the mice in GH group started to receive the IDP diet from GD0. Pregnant mice’s body weights and FPG levels were detected at GD0, GD3, GD6, GD9, GD12, GD15, GD18 respectively, in three groups. By the end of delivery, after 8 h fasting, dams (n = 10/group) were euthanized with 10% chloral hydrate and then killed by cervical dislocation.
Blood and Tissue Sample CollectionBefore the execution of the mice in different groups, whole blood was collected via orbital venous plexus at the indicated time points and then allowed it to clot. Serum samples were obtained by centrifuging at 3,000 rpm, 4°C for 10 minutes, then kept at –80°C. Thyroid, pancreas and liver tissues were harvested from the sacrificed mice after dissection. Major portions of thyroid and pancreas tissues were fixed in 4% paraformaldehyde solution for histological examination. Liver tissues were washed with saline and immediately frozen in liquid nitrogen, then stored at –80°C for western blotting and biochemical analysis.
Glucose tolerance testTo perform glucose tolerance test (GTT), mice were fasted 8 h after delivery (n = 8/group) and then the blood glucose from tail vein was measured by glucometer (t = 0) (ACCU-CHEK Performa, Roche, USA). At this point, glucose (2 g/kg of body weight) was injected intraperitoneally. Blood glucose samples were tested at 30, 60, 90, 120 and 150 min, respectively. Area-under-curve (AUC) was calculated by the trapezoid rule.
Plasma biochemical parametersThe plasma levels of total cholesterol (TC), triglycerides (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), and hepatic concentrations of TC, TG were determined (n = 10/group), respectively, according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). The levels of TSH, FT3, FT4 and Insulin in plasma were detected by ELISA kits (n = 10/group) (Jianglai, Shanghai, China). The plasma glucose levels were analyzed using a glucose analyzer (n = 10/group). The homeostatic model assessment of insulin resistance (HOMA-IR) index was calculated using the following formula: fasting insulin levels (mIU/L) × fasting glucose levels (mmol/L)/22.5.
Histological analysesLiver, thyroid and pancreas tissues were fixed in 4% paraformaldehyde solution and dehydrated in a graded series of ethanol, then embedded in paraffin and cut into 4 μm sections. The sections were deparaffinized with xylene, rehydrated in a descending graded series of ethanol and stained with hematoxylin and eosin (H&E) to assess morphological changes of the thyroid glands or pancreatic islets or hepatic cells, respectively.
Protein extraction and Western BlottingMurine livers were homogenized in ice-cold RIPA lysis buffer containing 1% phenylmethanesulfonyl fluoride (PMSF) and 2% phosphatase inhibitor cocktail, followed by centrifugation at 15,000 rpm, 4°C for 15 min to harvest the protein lysates. Total protein concentrations were determined by bicinchoninic acid (BCA) method. The equal amounts of proteins for each lane were aliquoted, denatured and then frozen at –80°C before experiment. Electrophoresis was conducted to separate protein samples using SDS-PAGE, which were further transferred onto PVDF membranes. The membranes were blocked with 5% skim milk for 1.5 h at room temperature, and incubated with appropriate primary antibody at 4°C overnight. After being washed four times with TBST buffer for 8 min/each, the membranes were incubated with secondary antibodies at room temperature for 1–1.5 h, and then the signals were detected using enhanced chemifluorescence reagent and the densities of the bands were quantified by scanning densitometry using Image Software. Each sample was analyzed by an average of three independent tests involving different gels. Antibodies including protein kinase B (AKT), phospho-protein kinase B (Thr308) (p-AKT T308), forkhead box transcription factor O1 (FoxO1), phospho-forkhead box transcription factor O1 (p-FoxO1), ATP citrate lyase (ACL), phosphoenolpyruvate carboxykinase (PEPCK), glycogen synthase kinase 3 beta (GSK3β) and phospho- glycogen synthase kinase 3 beta (p-GSK3β) were purchased from Cell Signaling Technology Inc. Antibodies against p-ACL, Sterol regulatory element binding protein-1c (SREBP1c), 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1), Carnitine palmitoyl transferase 1A (CPT1A), LDL receptor (LDL-R), Apolipoprotein AI (ApoA1), Apolipoprotein CⅢ (APOC3) were from Abcam. Glucose 6-phosphatase (G6Pase) was purchased from Thermo Scientific. Type I iodothyronine deiodinase (DIO1) was from Santa Cruz Biotechnology Inc.
Statistical analysisExperiment data were analyzed using SPSS 16.0 software (IBM Corporation, Armonk, USA) and expressed as mean ± SEM. Comparisons among groups were performed using one-way or two-way ANOVA, followed by Student-Newman-Keuls (SNK) multiple range test or Tukey’s multiple comparison test. The pregnancy rates were statistically processed by chi-square test of four-fold table. A value of p < 0.05 indicated a statistically significant difference. Graphics were constructed using GraphPad Prism 6 (GraphPad Software, San Diego, CA).
The whole experimental scheme was demonstrated as Fig. 1A. As expected, we found that serum levels of FT3 and FT4 were significantly decreased in the female mice after 28 days of feeding the IDP diet compared to those with the standard diet (p < 0.05, Fig. 1B). Meanwhile, the plasma TSH levels in PH group were markedly higher than those in GH and NC groups (p < 0.05, Fig. 1B). To induce gestational hypothyroidism, the GH group was treated with IDP diet from day 1 of pregnancy. After delivery, plasma FT3, FT4, and TSH levels were measured to confirm the success of the model. Like pre-pregnant hypothyroidism, gestational hypothyroidism also observably lowered serum levels of FT3 and FT4 while enhanced TSH levels in the GH group by the end of pregnancy as compared with the NC group (p < 0.05, Fig. 1C). Similarly, hematoxylin-eosin staining manifested that the mice fed with IDP diet showed the overt destruction of thyroid gland with conspicuous hypertrophic changes, which was connected with the hyperplasia of follicular cells and the decrease in follicular diameters and colloids. The HE staining data also indicated that the dams in GH and PH groups had the overall increased thickness of thyroid gland and the decreased mean size of follicular diameter as compared with those in NC group (Fig. 1D). More importantly, DIO1 plays a key role in TH activation, as shown in Fig. 1E, murine hepatic DIO1 protein levels have been markedly and equally inhibited in GH and PH groups as compared with those in NC group (p < 0.05).
Histopathological and hormonal examination of murine thyroid tissues revealed the hypothyroidism in postpartum dams
(A) Schematic timeline and treatments of current study. Female mice (C57BL/6J) were fed with iodine-deficient diet containing 0.15% propylthiouracil or with standard diet for 4 weeks starting at the age of week 5. (B) Free triiodothyronine (FT3), free thyroxine (FT4) and thyroid- stimulating hormone (TSH) levels were determined by ELISA kit respectively in the mice at week 9 (n = 6/group). After mating, the mice were either maintained the same diet feeding as pre-pregnancy (PH and NC groups) or switched to IDP diet feeding at day 1 of pregnancy (GH group) throughout the whole gestation. (C) The serum levels of FT3, FT4, and TSH in pregnant mice (n = 10/group) were analyzed by ELISA after delivery, respectively. (D) Paraffin-embedded murine thyroid tissues in various groups were stained with Hematoxylin and Eosin (H&E) dye. Morphological and pathological changes were observed by a microscope at ×200 in original magnification. Arrow: thyroid follicular cells. Scale bar 50 μm. (E) Hepatic protein expressions of DIO1 in dams in different groups were assayed by immunoblotting and quantified by actin levels. Data are presented as mean ± SEM, ** p < 0.01, *** p < 0.001. NC: normal control group, GH: gestational hypothyroidism group, PH: pre-pregnant hypothyroidism group.
Before pregnancy, the female mice in PH group grew slowly and showed a remarkable reduction in body weight after one-week administration of the IDP diet (Fig. 2A). On the contrary, hypothyroidism led to a significant increase in blood glucose levels under fasting conditions after two weeks of IDP diet feeding (Fig. 2B). Initially (GD0), the body weights of the pregnant mice were similar between GH and NC groups, which were, however, higher than those in PH group. After 18 days of gestation, the weight gains of dams in GH group fed with IDP diet during pregnancy were effectively inhibited as compared with those in the normal control group, but ultimately, PH group was revealed more powerful suppression of body weights than GH and NC groups (p < 0.05, Fig. 2C). At the same time, dams in PH group displayed a persistent hyperglycemia from GD0 to GD18, and those in GH group exhibited dramatically elevated blood glucose levels starting from GD9, as compared with NC group (Fig. 2D). Additionally, the female mice in PH group displayed a significant decrease in food intake (Fig. 2E). Like pre-pregnant hypothyroidism, the gestational hypothyroidism elicited the same degree of reduction on food intake in GH group, which was significantly lower than that in NC group (Fig. 2F). Moreover, the rates of pregnancy between control and PTU-treated animals were significantly different (Fig. 2G). In comparison with NC group, the litter sizes of GH and PH groups were markedly reduced, but there was no significant difference between GH and PH groups (p < 0.01, Fig. 2G).
Both pre-pregnant hypothyroidism and gestational hypothyroidism induced slow weight gains and hyperglycemia during gestation
Murine body weights (A) and fasting plasma glucose levels (B) in each group (n = 10) were recorded once a week from week 5 to week 9. Maternal body weights (C) and fasting glucose concentrations (D) were measured once every three days from GD0 to GD18 in different groups (n = 10). (E) The 24 h-food intakes in three groups were measured once a week from week 5 to week 9. (F) The 24 h-food intakes were detected once every three days from GD0 to GD18 in different groups. (G) The pregnancy rate and litter size of each group. Data are presented as mean ± SEM. & p < 0.05, &&& p < 0.001, PH group vs. NC group; # p < 0.05, ## p < 0.01, ### p < 0.001, PH group vs. GH group; + p < 0.05, +++ p < 0.001, GH group vs. NC group by two-way repeated-measure ANOVA with Tukey’s multiple comparison test. NC: normal control group, GH: gestational hypothyroidism group, PH: pre-pregnant hypothyroidism group.
To compare the effects of pre-pregnant hypothyroidism and gestational hypothyroidism on lipid metabolism in dams after delivery, we analyzed the levels of TG, TC, LDL-C, HDL-C in serum and the contents of TG and TC in murine livers. Plasma TG levels were vastly reduced in GH and PH groups compared with those in NC group, in which there was no significant difference between GH and PH groups (p < 0.05, Fig. 3A). Upon comparison with normal controls, plasma TC and LDL-C levels as well as hepatic TG levels were markedly raised in PH group. Moreover, the three biochemical indexes in GH group were much more elevated than those in PH group, suggesting more significant lipid dysregulation in dams fed with IDP diet starting from pregnancy (p < 0.05, Fig. 3B, 3C and 3E). Nevertheless, the plasma HDL-C was revealed the same levels in all groups (p > 0.05, Fig. 3D). Compared with the NC group, the hepatic TC levels in the GH and PH groups were equally and observably increased (p < 0.05, Fig. 3F). By H&E staining, the murine liver tissues in NC group showed normal hepatic cells with clear and distinct nuclei and cytoplasm, while those in GH group manifested with numerous spherical vacuoles of fat droplets at the periphery of hepatic lobule. In comparison with GH group, the liver tissues from PH group displayed vacuoles of fat droplets with decreased size and number (Fig. 3G).
Gestational hypothyroidism induced more prominent plasma TC and hepatic TG dysregulation than pre-pregnant hypothyroidism in dams
Serum and liver samples were collected after an 8-h fast from hypothyroid or normal mice at the end of experiment. (A-D) Serum TG, TC, LDL-C and HDL-C levels were measured, respectively. The TG (E) and TC (F) contents in murine livers were determined, respectively. (G) Liver tissues from hypothyroid or normal mice were fixed with 4% paraformaldehyde for H&E staining. The results are shown at ×200 magnification. CV: central vein. Arrow: fat droplets. Scale bar 50 μm. Data are presented as the mean ± SEM (n = 10/group), * p < 0.05, ** p < 0.01, *** p < 0.001. NC: normal control group, GH: gestational hypothyroidism group, PH: pre-pregnant hypothyroidism group.
In order to explore whether the dysregulated lipid metabolism in dams was owing to altered protein expressions in murine livers, we respectively detected the protein levels of SREBP1c, p-ACL, HMGCS1 and CPT1A. Our data demonstrated that the SREBP1 protein levels and the ratios of p-ACL to ACL were markedly increased in PH and GH groups as compared to the normal control group, while the enhancement elicited by gestational hypothyroidism was substantially sharper than that in PH group, indicating more effective promotion of hepatic lipogenesis in dams in GH group (p < 0.05, Fig. 4A and 4B). Furthermore, CPT1A protein levels were moderately suppressed in PH group compared to NC group, whereas the levels in GH group were showed much more inhibition than those in PH group (p < 0.05, Fig. 4C), suggesting much more accumulation of hepatic fat in GH group. Subsequently, we examined the protein expression of HMGCS1, which was greatly and equally raised in two hypothyroid groups as compared with NC group (p < 0.05, Fig. 4D). In addition, more significant suppression of hepatic LDL-R level was observed in mice in GH group as compared with PH group (p < 0.05, Fig. 4E). It should be pointed out that the hepatic ApoA1 protein levels were similar in all groups, which matches up with plasma HDL-C data (p < 0.05, Fig. 4F). Additionally, the APOC3 protein levels have been observably restrained in two hypothyroid groups (p < 0.05, Fig. 4G), which was consistent with the data in Fig. 3A.
Gestational hypothyroidism more effectively promoted hepatic fatty acid synthesis than did the pre-pregnant hypothyroidism in dams
(A) The murine hepatic protein levels of SREBP1c in NC, GH, PH groups were determined by immunoblotting and quantified by actin levels. (B) The protein levels of ACL and p-ACL were displayed by immunoblotting, and the phosphorylated ACL was quantified by total ACL. (C-G) Hepatic protein expressions of CPT1A, HMGCS1, LDL-R, ApoA1, APOC3 in different groups were assayed by immunoblotting and quantified by actin levels, respectively. Data are presented as the mean ± SEM from 3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001. NC: normal control group, GH: gestational hypothyroidism group, PH: pre-pregnant hypothyroidism group.
The serum insulin levels from the hypothyroid and euthyroid groups did not differ significantly, whereas the hypothyroid mice exhibited a significant increase in fasting plasma glucose, which led to higher HOMA-IR values than those in the control mice. Of note, the values of HOMA-IR were equally enhanced in two hypothyroid groups (p < 0.05, Fig. 5A-5C). As presented in Fig. 5D, the fasting plasma glucose levels were similar between GH and PH groups, which were, however, observably higher than those in normal control group. For the glucose tolerance tests, we can see that after administration of glucose in dams after delivery, both of the induced glucose disposals in PH and GH groups were sharply impaired as compared to normal controls. The dams from two hypothyroid groups displayed the same degree of increase in the AUC of blood glucose by the GTT as compared with NC mice (p < 0.05). By H&E staining, the murine islets in NC group appeared pale with stained rounded or oval areas inside the pancreatic lobules surrounded by the exocrine parenchyma (Fig. 5E). While pancreatic tissues from two groups of hypothyroid mice showed severe degeneration of islets, with the irregular shape and obvious reduction in the size and number, in which many of the islet cells demonstrated clear or vacuolated cytoplasm and pyknotic nuclei. Given the aforementioned changes of those characteristics, we examined the phosphorylated (p-) forms of AKT Thr308 and the FoxO1 to explore the intrahepatic mechanism of insulin resistance. We found that the ratios of phosphorylated AKT (p-AKT T308) to total AKT indicated the effective inhibition of AKT activity elicited by the two hypothyroidism types as compared to that in the NC group. However, no difference of AKT activities was observed between two hypothyroidism groups (p < 0.05, Fig. 5F). The same pattern of effect on phosphorylated FoxO1 was also revealed in the mice from two hypothyroid groups. The ratios of p-FoxO1 to total FoxO1 were dramatically reduced in GH and PH groups, elucidating the similar impairment for hepatic insulin signaling in dams treated with IDP diet starting before and during pregnancy (p < 0.05, Fig. 5G).
Gestational hypothyroidism significantly impaired hepatic insulin sensitivity on dams, which was similar to the effect by pre-pregnant hypothyroidism
Murine plasma insulin levels (A) and blood glucose (B) were detected after 8 h fast (n = 10/group). (C) The homeostasis model assessment of insulin resistance (HOMA-IR) was used as a screening index for insulin sensitivity and calculated via the values measured in (A) and (B). (D) Blood glucose levels during a glucose tolerance test (GTT, 2 g/kg glucose) were assayed after delivery (n = 8/group). (E) Pancreatic tissues from hypothyroid or normal mice were fixed in 4% paraformaldehyde for H&E staining. The results are shown at ×200 magnification. Arrow: pancreatic islet. Scale bar 50 μm. (F) Displayed are the protein levels of AKT, and p-AKT S473. The AKT Ser473 phosphorylation was quantified by total AKT. (G) Total and phosphorylated forms of FoxO1 were detected by immunoblotting, and the p-FoxO1 was quantified by total FoxO1. All data are presented as the mean ± SEM. & p < 0.05, && p < 0.01, &&& p < 0.001, PH group vs. NC group; + p < 0.05, ++ p < 0.01, +++ p < 0.001, GH group vs. NC group by two-way repeated-measure ANOVA with Tukey’s multiple comparison test. *** p < 0.001. NC: normal control group, GH: gestational hypothyroidism group, PH: pre-pregnant hypothyroidism group.
Both PEPCK and G6Pase are key rate-limiting enzymes required for hepatic gluconeogenesis, which plays an important role in maintaining glucose homeostasis in fasting conditions, and which is also dramatically enhanced under some pathological conditions such as insulin resistance. Hence, we next examined the two protein expression levels in murine livers by western blotting to assess the effects of two different hypothyroidisms on dams. Fig. 6A and 6B suggest that the PEPCK and G6Pase protein levels have been dramatically elevated in GH and PH groups as compared with NC group (p < 0.05), but those levels in two hypothyroid groups did not differ statistically (p > 0.05). On the other hand, the ratios of p-GSK3β to total GSK3β were remarkably curbed in two hypothyroid groups, illustrating improved hepatic GSK3β activity or weakened hepatic glycogen synthesis in two hypothyroid mice (p < 0.05, Fig. 6C).
Pre-pregnant Hypothyroidism and gestational hypothyroidism vastly and equally enhanced the hepatic gluconeogenesis and weakened glycogen synthesis in dams
The PEPCK (A) and G6Pase (B) protein levels in the murine livers from different groups were examined by immunoblotting and the results were quantified by β-actin, respectively. (C) Total and phosphorylated forms of GSK3β were detected by immunoblotting, and the p-GSK3β was quantified by total GSK3β. (D) TH regulates lipogenesis or lipolysis via several key proteins such as SREBP1c, ACL, CPT1A and HMGCS1, respectively. TH increases cholesterol uptake into the liver by inducing the protein expression of LDL-R. Insulin signaling inhibits GSK3β to induce glycogen synthesis and reduce the transcription of gluconeogenic enzymes by inactivating FoxO1. Data are presented as the mean ± SEM from 3 independent experiments. ** p < 0.01, *** p < 0.001. NC: normal control group, GH: gestational hypothyroidism group, PH: pre-pregnant hypothyroidism group.
Based on our present study, both pre-pregnant hypothyroidism and gestational hypothyroidism, as compared with euthyroid controls, led to decreased growth and hyperglycemia in mice. On the contrary, hypothyroidism is associated with weight gains elicited by reduced energy expenditure in humans. In spite of the difference, our current findings regarding the inhibited growth are supported by the previous studies in experimental rodents with hypothyroidism [29, 30]. In comparison with NC group, the female mice in hypothyroid groups displayed a significant decrease in food intake (Fig. 2E and 2F), it may explain the differences of body weights exerted by hypothyroidism between humans and rodents.
The patients with hypothyroidism have been associated with elevated serum TC and TG levels as well as NAFLD [31]. However, our current results showed that two hypothyroidism groups elicited significantly reduced plasma TG (Fig. 3A), which may be partly explained by both significantly increased serum pseudocholinesterase (PChE) and lipoprotein lipase (LPL) activity elicited by TH deficiency in animals, although the activities of both enzymes are reduced in hypothyroid patients [32]. Moreover, APOC3, an inhibitor of LPL activity, plays a crucial role in the catabolism of triglycerides, and the suppressed expression of the gene may be connected with the raised LPL activity and then the decrease in plasma TG level [33], which is consistent with our current result (Fig. 4G).
Unexpectedly, our findings indicated that gestational hypothyroidism may be more potent than pre-pregnant hypothyroidism to increase plasma TC and LDL-C levels in dams, which may be owing to intrahepatic mechanisms. The GH may be more effective to decrease the hepatic absorption of plasma cholesterol by reducing the number and activity of hepatic LDL-R, than does the PH, contributing to the difference of plasma TC, LDL-C levels in two hypothyroid groups.
In our current study, in addition to the decrease in serum TH, hepatic DIO1, a key enzyme that activates TH and converts thyroxine to triiodothyronine, is also downregulated in GH and PH groups, which matches up with previous study [34, 35]. The mechanism for the regulation of SREBP1c by TH has remained controversial. One experimental animal study reported that SREBP1c transcription was negatively regulated by TH via a putative negative thyroid hormone response element (nTRE) [36], but another study has shown that SREBP1c transcription is upregulated by non-genomic thyroid hormone signaling [37]. In our current study, the expressions of some hepatic TH-responsive proteins were sharply enhanced, such as SREBP1c, ACL [38], HMGCS1 [9], which are consistent with the promotions of fatty acid and cholesterol synthesis, respectively (Fig. 4A, 4B and 4D). Whereas CPT1A, another TH- responsive protein [39], was more greatly down-regulated in GH group than did in PH group (Fig. 4C), that matches with more accumulation of hepatic TG in GH mice. To date, no systematic researches have been conducted to explore the time effects of thyroid hormone on lipid dysregulation. Hence, why the GH group showed unexpected more vigorous elevation of hepatic lipids than PH group requires further investigation.
TH is one of the key regulators of development, growth and metabolism in all tissues. It has been reported that lessened TH levels could have a role in progression of pancreatic β-cell dysfunction, resulting in the decrease in glucose stimulated insulin secretion [40, 41], which is consistent with the pathological changes of the pancreas in our present study (Fig. 5E).
Due to the influence of many factors, our findings may suggest that two types of hypothyroidism equally and significantly impaired hepatic insulin sensitivity, and that these results were achieved by regulating the key proteins involved in the insulin signaling, such as AKT, FoxO1 (Fig. 5F and 5G). Meanwhile, enhanced fatty acids delivery to the liver increases hepatic acetyl-CoA (Ac-CoA) content, leading to allosteric activation of pyruvate carboxylase (PC) that promotes hepatic gluconeogenesis [42]. PEPCK and G6Pase are involved in the rate-limiting steps in gluconeogenesis, and it has been recently reported that TH is essential for PEPCK and G6Pase transcriptions [43]. In this study, the up-regulations of PEPCK and G6Pase were observed in hypothyroid mice, which might be mediated by the indirect action of TH (Fig. 6A and 6B). In addition, it is well accepted that the repression of AKT phosphorylation downregulates GSK3β phosphorylation [44]. Our current study manifested that hypothyroidism in two groups of mice elicited the same degree of promotion of GSK3β activity, thus resulting in the same inhibition of hepatic glycogen synthesis, which is consistent with our data that the FPG levels in hypothyroid mice were enhanced (Fig. 5B and 6C).
In conclusion, our current study illuminated that mild hypothyroidism may have an impact on insulin resistance and result in metabolic dysfunction of glucose and lipid in dams. More importantly, our present results revealed that gestational hypothyroidism may elicit much more significant lipid dysregulation in dams than do pre-pregnant hypothyroidism. These findings may shed a new light on future clinical practice.
Keyang Chen and Zhengxuan Jiang designed the study, obtained research fund, supervised the study and reviewed/revised the manuscript. Jun Zhou and Xuan Dong performed the experiments, analyzed and interpreted data, and wrote the draft of manuscript. Yajing Liu, Yajing Jia and Yang Wang prepared reagents and other experimental materials. Ji Zhou validated the data. All authors read and approved the manuscript for publication.
This work was supported by a grant from the National Natural Science Foundation of China (NSFC, 81570786 to KC) and Wanjiang young scholar grant from Anhui Province of China (9101041203 to Z.J).
All authors declare no conflict of interest.
SREBP1c, Sterol regulatory element binding protein-1c; p-ACL, phosphorylated ATP citrate lyase; CPT1A, Carnitine palmitoyltransferase A; LDL-R, low-density lipoprotein cholesterol receptor; p- AKT, phosphorylated protein kinase B; p-FoxO1, phosphorylated forkhead box transcription factor O1; APOC3, Apolipoprotein CⅢ.
