3.1. HFD Feeding Induced Obesity and Altered Metabolic Syndrome
Previous studies suggested that consumption of a high-fat diet (HFD) rapidly reprograms systemic metabolism and, particularly, causes obesity [
38,
39]. Here, the effect of HFD on mice, after Kunming mice were fed by HFD or normal chow (NCW) for 13 weeks was evaluated.
Figure 1A–C shows body weight, body mass index (BMI) and whole body fat mass were significantly higher in HFD-fed mice than NCW-fed mice. Further analysis showed that HFD-fed mice gained more inguinal fat (
Figure 1D), gonadal fat (
Figure 1E) and perirenal fat (
Figure 1F), all of which were significantly increased when compared to NCW-fed mice, as reported also by Jeffery et al. [
40]. Furthermore, consistent with previous findings that the expansion of adipose tissues in overweight or obese humans induced inflammation [
41,
42,
43], qRT-PCR analysis showed that genes involved in promoting inflammation were expressed at high levels in adipose tissues of HFD-fed mice (
Figure 1G). All results indicate that HFD feeding induced obesity.
Interestingly, it was observed that HFD feeding significantly increased accumulation of intramyocellular lipids as compared to NCW feeding (
Figure 1H). Recently, it was demonstrated that the level of intramyocellular lipids can be used as a marker of insulin resistance, especially in type 2 diabetes mellitus [
44]. Therefore, glucose levels and insulin sensitivity in HFD- and NCW-fed mice were evaluated subsequently.
Figure 1I shows the higher levels of fasting blood glucose (at 0 min) were observed in HFD-fed mice. Glucose-tolerance tests (GTTs) show that glucose levels increased more in HFD-fed mice than NCW-fed mice (
Figure 1I). Similarly, higher glucose levels at 0, 15, 30, 60, and 90 min of insulin-tolerance tests (ITTs) were found in HFD-fed mice, indicating that a HFD impaired glucose intolerance and decreased insulin sensitivity (
Figure 1J). Taken together, these results demonstrate that a HFD feeding could induce obesity and alter metabolic syndrome, such as increasing white fat (WAT) production, lipid accumulation in muscle, and insulin resistance.
3.2. Betaine Supplementation Inhibited White Fat Production in HFD-Induced Obese Mice
Obesity is a major driver of some metabolic diseases, but there are no effective drugs to treat obesity. Recently, Ejaz et al. [
32] reported that betaine could decrease inguinal WAT of mice fed with HFD, prompting the hypothesis that betaine supplementation can be considered for treatment of obesity. First, evaluation of the effect of betaine supplementation on adipocytes development, after mice fed with HFD or NCW were treated with or without 1% betaine in water was performed. This dose of betaine was intentionally chosen so as to align with previous studies that showed improved glucose homeostasis and prevention of fatty liver induced by a high-fat diet [
32,
45].
Figure 2A,B, in agreement with a previous report [
32], shows betaine supplementation had little influence on the weight of NCW-fed mice, but significantly reduced body weight gain of HFD-fed mice. Expectantly, both HFD- and NCW-fed mice exhibited decreased inguinal (
Figure 2C,D), gonadal (
Figure 2E,F), as well as total fat mass (
Figure 2G,H), when HFD- and NCW-fed mice were fed with 1% betaine in water, respectively. However, in contrast to the fact that 1% betaine supplementation significantly reduced perirenal fat mass in HFD-fed mice, 1% betaine supplementation had little influence on perirenal fat mass in HFD-fed mice (
Figure 2I,J). Notably, in line with the observed resistance to HFD, 1% betaine supplementation displayed significant changes in plasma lipid and lipoprotein levels in HFD-fed mice. These include a significant decrease in plasma alanine transaminase (ALT) (
Figure 2K), aspartate aminotransferase (AST) (
Figure 2L), triglycerides (TG) (
Figure 2M), cholesterol (TC) (
Figure 2N) and low-density lipoprotein (LDL) (
Figure 2O), and in contrast, a slight increase in plasma high-density lipoprotein (HDL) (
Figure 2P). Meanwhile, we found that 1% betaine supplementation had no significant effect on plasma ALT, AST, LDL, HDL, TG and TC in NCW-fed mice compared to the control (
Figure 3), which is consisted consistent with previous results [
46,
47]. Taken together, these results suggest that betaine supplementation in mice could inhibit WAT formation in vivo.
Increased newly adipose tissue mass is ascribed to pre-adipocytes proliferation and differentiation, and the hypertrophy of mature adipocytes [
48]. To explore whether betaine supplementation inhibits WAT formation through modulating pre-adipocytes proliferation and differentiation, 3T3-L1 white pre-adipocytes were treated with 20 mM betaine. First, CCK-8 and EdU assays were performed to evaluate the effect of betaine on the proliferation of 3T3-L1 white pre-adipocytes. As shown in
Figure 4A, CCK8 analysis showed betaine treatment reduced the number of 3T3-L1 white pre-adipocytes, when compared to the untreated group. Furthermore, these results were further confirmed by an EdU assay. The ratio of EdU positive cells indicated the cell in DNA synthesis phage.
Figure 4B shows the ratio of EdU positive cells was decreased remarkably after betaine treatment, suggesting that betaine might inhibit the proliferation of 3T3-L1 white pre-adipocytes. P21 is a cell cycle-arrest regulator, which is downstream of P53 [
49,
50]. Cyclin D/E are mammalian G1 cyclins that are both required and rate limiting for entry into S phase, and inhibition of cyclin D/E function can induce cell cycle arrest [
51,
52]. Agreeing with these observations, qRT-PCR analysis shows that betaine treatment caused an increase in the expression level of P21 and P53, while inhibiting cyclin D/E expression (
Figure 4C). These results indicate that betaine might inhibit the proliferation of 3T3-L1 white pre-adipocytes by regulating cell cycle regulators. Subsequently, investigation of the effect of betaine on the differentiation of 3T3-L1 white pre-adipocytes were performed. The results showed that betaine treatment significantly reduced the number of oil red O+ cells (
Figure 4D), triglyceride accumulation (
Figure 4E), when compared to the control. C/EBPa and PPARγ, are two key transcription factors, which can mediate adipocyte differentiation and hypertrophy by regulating adipogenic gene expression [
53,
54]. Wu et al. [
55] reported that mice deficient in C/EBPα have defective development of adipose tissue, for instance. Jones et al. [
56] suggested that deletion of PPARγ in adipose tissues of mice protects against high fat diet-induced obesity lipid accumulation. Expectantly, qRT-PCR analysis suggested that C/EBPa and PPARγ were remarkably suppressed during adipocytes differentiation after betaine treatment (
Figure 4F), suggesting that betaine could inhibit the differentiation of 3T3-L1 white pre-adipocytes, which is consistent with betaine being used as a dietary supplement in pig nutrition to reduce fat deposition [
57]. Therefore, the above results confirm that inhibiting the proliferation and differentiation of pre-adipocytes could be considered as a reason that betaine supplementation decreases WAT formation.
Mature WAT contains most of the energy stored in the form of triglycerides (TGs), however, the potential mechanisms of how betaine consume mature WAT induced by HFD is still unclear. Recently, Lee et al. [
58] reported that betaine treatment leads to an upregulation of mitochondrial respiration and cytochrome c oxidase activity in H2.35 cells. Surprisingly, estimated mitochondrial content by measuring the mtDNA/nDNA ratio, found that 1% betaine supplementation significantly increased the relative content of mitochondria in WAT of mice fed with HFD (
Figure 4G). Moreover, in accordance with a previous report [
32], the transcript levels of brown adipocyte markers were increased in WAT (
Figure 4H), after mice fed with HFD were treated with 1% betaine in water. Mitochondria provide the majority of cellular energy in the form of ATP through oxidative phosphorylation (OXPHOS) [
59]. Previously, numerous studies have revealed that WAT has fewer mitochondria [
14,
60]. Conversely, brown adipose tissue (BAT) contains dense mitochondria. Particularly, in response to an energy imbalance, BAT robustly enhances the whole animal energy expenditure in the form of heat [
60,
61,
62]. Loss of BAT function is linked to obesity and metabolic diseases [
63,
64]. These data in this study suggest that betaine supplementation might consume WAT induced by HFD by stimulating mitochondrial biogenesis in WAT and promoting browning of WAT.
Taken together, the above findings indicate that preventing the formation of new WAT and guiding the original WAT burning, betaine supplementation inhibited WAT production in HFD-induced obese mice.
3.3. Betaine Supplementation Decreased Intramyocellular Lipid Accumulation in HFD-Induced Obese Mice
Obesity leads to accumulation of ectopic fat such as intrahepatic lipids and intramyocellular lipids [
19,
65]. Several previous results showed that betaine reduces hepatic triglyceride content [
45]. To further evaluate the effect of betaine on HFD-induced obesity, the authors investigated whether betaine supplementation prevents accumulation of intramyocellular lipids, which is induced by HFD.
Figure 5A is consistent with previous findings; intramyocellular lipid content positively correlated with body weight. It was found that 1% betaine supplementation prevented intramyocellular lipid accumulation induced by HFD (
Figure 5B,C). Fatty acid is a main substrate of lipid metabolism. To confirm whether betaine reduced intramyocellular lipid accumulation induced by HFD is associated with fatty acid, comparison of fatty acid composition in muscle from HFD-induced mice treated with and without 1% betaine in water was performed.
Figure 5D shows 1% betaine supplementation just increased a part of saturated fatty acids (SFA; C15:0 and C18:0), monounsaturated fatty acids (MUFA; C18:1n9c and C20:1), which is consistent with a previous study that found betaine supplementation could slightly increase content of SFA and MUFA in muscles from an obese pig breed [
45]. Previously, some studies have proposed that SFA length and the physical characteristics of the triacylglycerol structure of SFA may impact lipoprotein metabolism [
66]. These findings are a reminder that betaine supplementation might mediate lipid metabolism by regulating fatty acid composition in muscle from HFD-induced mice. Interestingly, further analysis found that almost all polyunsaturated fatty acids (PUFA) in intramyocellular lipids were significantly increased in muscle tissues, when HFD-fed mice were fed with 1% betaine (
Figure 5D). This finding strongly confirmed that betaine supplementation could alter fatty acid composition in muscle of HFD-induced obese mice to mediate adipocytes development, because previous studies have demonstrated that PUFA mediates adipocyte proliferation, differentiation and energy metabolism by regulating the expression levels of genes related to lipid metabolism [
67,
68,
69]. Particularly, PUFA represses fatty acid synthesis by decreasing the expression of SREBP-1c, whereas it enhances fatty acid oxidation by activating PPARα [
70,
71]. The authors thus evaluated whether betaine supplementation could regulate the synthesis and oxidation of fatty acid in intramyocellular lipids of HFD-fed mice. A qRT-PCR analysis showed that 1% betaine supplementation up-regulated PPARα expression (
Figure 5E) but down-regulated SREBP-1c expression (
Figure 5F). Conformably, 1% betaine supplementation enhanced genes associated with fatty acid oxidation (
Figure 5E), whereas inhibited genes were involved with fatty acid synthesis (
Figure 5F), suggesting that, by increasing PUFA to promote fatty acid oxidation but inhibit synthesis, betaine supplementation might reduce intramyocellular lipid accumulation in HFD-induced obese mice. Taken together, the above results indicate that, by mediating fatty acid metabolism, betaine supplementation reduced intramyocellular lipid accumulation in HFD-induced obese mice.
3.4. Betaine Supplementation Relieved Inflammation and Improved Insulin Resistance in HFD-Induced Obese Mice
Insulin resistance is a common characteristic associated with obesity [
72]. To confirm that betaine supplementation limited HFD-induced obesity, a GTT and ITT to evaluate insulin sensitivity in HFD-induced obese mice fed with and without betaine in water.
Figure 6A,B demonstrates that 1% dietary betaine supplementation reduced values in insulin-tolerance tests (ITTs), whereas it improved glucose metabolism in glucose-tolerance tests (GTTs) in HFD-induced mice, as compared to the control group. Consistently, the change of glycemia levels on the ITTs were further evaluated as percentage, and found that the percent of decrease glycemia is greater in experimental group than control group, indicating that betaine supplementation may improve insulin sensitivity in HFD-induce obese mice (
Figure 6C). Subsequently, to further confirm these results in vivo, an insulin-resistant adipocyte model according to the method described previously by Xu et al. [
73] was established.
Figure 6D shows betaine treatment (20 mM) increased glucose uptake by measuring the glucose levels of medium cultured insulin-resistant 3T3-L1 cells, suggesting that betaine treatment improved insulin resistance in an insulin-resistant adipocyte model. Previously, some studies revealed that obesity-induced inflammation, especially in obese adipose tissue, is an important cause of obesity-induced insulin resistance [
42,
74,
75,
76]. The rise in FFAs levels lead to proinflammatory gene expression and decreases sensitivity to insulin [
44,
77,
78]. Consistent with betaine supplementation improving insulin resistance, it also was observed that 1% betaine supplementation decreased the level of serum free fatty acids (FFAs) in HFD-fed mice (
Figure 6E), a result consistent with previous reports [
79]. Meanwhile, 1% betaine supplementation caused a significant decrease in the expression levels of some inflammatory stress-related factors in adipose tissues of HFD-fed mice (
Figure 6F), which has been reported in human adipocytes where betaine reduced hypoxia-induced expression of inflammatory adipokines [
80]. These results demonstrate that betaine supplementation improves insulin resistance in HFD-induced obese mice.
Increased insulin resistance causes hyperglycemia, which is a major metabolic abnormality in a great majority of patients with type 2 diabetes. To examine the possibility of dietary betaine supplementation in the treatment of hyperglycemia, mice were injected with streptozotocin (STZ), which induces hyperglycemia and even diabetes in vivo.
Figure 6G shows STZ successfully induced hyperglycemia. The STZ diabetic mice were then treated with betaine in water for 30 days and the blood glucose levels were found to decrease. Taken together, these data provide evidence that dietary betaine supplementation might improve obesity and non-obesity induced insulin resistance.