Results and discussion
HCFD combined with STZ effectively induces T2DM rats
The HCFD combined with the low-dose STZ-induced diabetes animal models have been recognized to mimic human T2DM for experimental purposes.14 In this study, after 6 weeks of induction, the levels of serum glucose and insulin were significantly higher in rats than those observed previously (p<0.001; figure 1A,B). In rats, the insulin resistance index was significantly higher than that previously recorded (p<0.001, figure 1C), indicating that insulin resistance had occurred in rats, which is a typical symptom of type 2 diabetes.
Figure 1Changes in type 2 diabetes mellitus rats reduced by high-carbohydrate and fat diet combined with streptozotocin. (A) Concentration of serum glucose before and after induction (n=16). (B) Changes in serum insulin before and after induction (n=8). (C) Insulin resistance index calculated by the values of serum glucose and insulin (n=8). (D,E) Changes in oral glucose tolerance test before and after induction (n=13). (F,G) Change in pyruvate tolerance test before and after induction (n=13). ***P<0.001. AUC, area under the curve; FBG, fasting blood glucose; HOMA-IR, homeostatic model assessment of insulin resistance index.
The OGTT is a glucose stress test used to understand islet β-cell function and the body's ability to regulate blood glucose. It is a specific test for diagnosing diabetes and is widely used in clinical practice. As we compared the OGTT between before and after induction, the results demonstrated that the glucose tolerance in rats was significantly reduced (p<0.001; figure 1D,E). Pyruvate is a substrate for gluconeogenesis, and PTT is commonly used to measure gluconeogenesis in the liver. Compared with the values before induction, gluconeogenesis in the liver of T2DM rats was severely impaired (p<0.001; figure 1F,G). The pyruvate injection failed to induce significant changes in the blood glucose levels.
The aforementioned results implied that rats presented apparent clinical symptoms of T2DM 6 weeks after the induction by using HCFD combined with a low dose of STZ. Hence, T2DM rats induced by HCFD combined with a low dose of STZ are valid and reliable.
Hb lesions effectively reduce blood glucose and serum insulin levels and improve insulin resistance in T2DM rats
To determine the role of Hb in the pathophysiology of diabetes, we first observed the effects of Hb lesions on serum glucose and serum insulin in T2DM rats. The results demonstrated that both the concentration of serum glucose and insulin in the lesion group were lower than those observed in the sham group (figure 2A,B). In T2DM rats, the insulin resistance index dramatically reduced after Hb lesions (figure 2, p<0.05). Based on these results, we determined that Hb lesions lower the blood glucose level and improve insulin resistance in T2DM rats. These results were consistent with the OGTT results (figure 2D,E). Hb lesions enhanced glucose tolerance and improved insulin resistance in T2DM rats. However, the mechanisms by which these results are achieved remain unclear. To this end, we examined the effects of Hb lesions on the liver, an essential metabolic organ.
Figure 2Effects of habenular nucleus lesions on the T2DM rats. (A) Changes in fast serum glucose in the sham and lesion groups of T2DM (n=8). (B) Serum insulin changes of sham and lesion groups of T2DM (n=8). (C) Insulin resistance index of sham and lesion groups of T2DM (n=8). (D,E) Oral glucose tolerance test of sham and lesion groups of T2DM rats (n=6.) (F,G) Pyruvate tolerance test of sham and lesion groups of T2DM (n=6). (H) Western blot of Akt, P-Akt, and the statistic results of Akt in four groups (n=3). (I) Statistic results of P-Akt (n=3). (J) Ratio of P-Akt and Akt (n=3). *P<0.05, **P<0.01, ***P<0.001. AUC, area under curve; FBG, fasting blood glucose; HOMA-IR, homeostatic model assessment of insulin resistance index; INS, fast serum insulin; N.S., no significance; T2DM, type 2 diabetes mellitus.
Akt is a substrate in the insulin signaling pathway, with insulin exerting glycemic regulation by activating Akt phosphorylation.16 17 Therefore, the degree of Akt phosphorylation is also an indicator of insulin sensitivity. Our results demonstrated that the levels of Akt in the liver of rats was not significantly altered, but the levels of phosphorylated Akt in the liver of T2DM rats were lower than that observed in the control, and the levels of phosphorylated Akt in Hb lesion group were higher than that of the sham (figure 2H,I). The ratio of phosphorylated Akt to Akt in the liver of the Hb lesion group was higher than that of sham (figure 2J). These results suggest that Hb lesions could promote Akt phosphorylation and improve insulin sensitivity in T2DM rats.
Hb lesions increase AMPK phosphorylation and upregulate IRs in the liver
The liver is the main organ that regulates glucose metabolism. In the liver cells, activation of AMPK can inhibit gluconeogenesis and glycogen synthesis.18 19 Phosphorylation of the AMPK α (AMPKα) subunit is the primary form of AMPK activation. Western blotting demonstrated that, although the total amount of AMPKα was not significantly different in the liver of all four groups (figure 3A), AMPKα phosphorylation (P-AMPKα) levels in T2DM rats were lower than those observed in the control, whereas P-AMPKα levels in the Hb lesions group were significantly higher than those in the sham group (figure 3B). Moreover, in the Hb lesion group, the ratio of P-AMPKα to AMPKα increased significantly (figure 3C). Consistent with AMPK, the two key rate-limiting enzymes of gluconeogenesis, glucose-6-phosphatase (p<0.001, figure 3D) and phosphoenolpyruvate carboxy kinase (p<0.001, figure 3E), were increased in the T2DM group and significantly decreased in the Hb lesion group. In T2DM rats, Hb lesions resulted in the inhibition of hepatic gluconeogenesis. PTT results further demonstrate that Hb lesions inhibit hepatic gluconeogenesis (p<0.01; figure 2F,G).
Figure 3Effects of habenular nucleus lesions on glucose metabolism in the liver of T2DM rats. (A) Western blot of AMPK, P-AMPK, and the statistic results of AMPK in four groups (n=6). (B) Statistic results of P-AMPK (n=6). (C) Ratio of P-AMPK and AMPK (n=6). (D) Concentrations of G6pase (n=6). (E) Concentrations of PEPCK in the liver of rats (n=6). (F) Concentration of glucagon in the liver (n=6). (G) Western blot and expression levels of the IR in the liver of rats (n=8). (H) Western blot and statistic results of the GR in rat liver (n=8). *P<0.05, ***P<0.001. AMPK, adenosine 5’-monophosphate-activated protein kinase; Ctrl, control; G6pase, glucose-6-phosphatase; GR, glucocorticoid receptor; IR, insulin receptor; P-AMPK, phosphorylated AMP-activated protein kinase; PEPCK, phosphoenolpyruvate carboxylated kinase; T2DM, type 2 diabetes mellitus.
In the liver, the upregulation of IRs results in the inhibition of hepatic glucose production by inhibiting glycogenolysis.20 In this study, we observed that the expression levels of the IR were lower in the liver of T2DM rats than in the control, Hb lesions increased the expression of IRs (figure 3G). Hb lesions improved insulin function by upregulating IRs and promoting Akt phosphorylation (figure 2I). Additionally, we observed no significant differences in the liver glucocorticoid receptor expression levels in the four groups, although they tended to be higher in the T2DM group than that in the control group and lower in the lesion group than in the sham group (figure 3H). Our results suggest that Hb lesions inhibit hepatic glycogen synthesis and glycogenolysis by upregulating the insulin signaling pathway. Glucagon is a hormone that antagonizes insulin and raises blood glucose levels. It can increase blood glucose by promoting glycogenolysis and gluconeogenesis. However, we failed to observe that Hb lesions could reduce glucagon in the liver of T2DM rats (figure 3F).
The aforementioned results suggest that Hb lesions inhibit gluconeogenesis and glycogenolysis by activating the AMPK signaling pathway and upregulating IRs.
Hb lesions improve the functions of the hippocampus and inhibit the sympathetic nervous system
BDNF is an essential member of the neurotrophin family and plays a crucial role in regulating the survival, growth, and maintenance of neurons. Reportedly, BDNF and its receptor TrkB levels are reduced in the hippocampus of diabetic mice.21 This was consistent with our results, demonstrating lower BDNF and TrkB levels in the hippocampus of T2DM rats than those observed in the control group (p<0.05; figure 4A,B). Studies have shown that reduced BDNF expression in the hippocampus of diabetic rodents have a disinhibited effect on the HPA axis.22 We observed that Hb lesions can significantly increase the expression of BDNF and TrkB in the hippocampus of T2DM rats (p<0.05; figure 4A,B), indicating that Hb lesions might inhibit the HPA axis in T2DM rats by increasing the expression of BDNF in the hippocampus. Although the glucocorticoid receptor expression in the rat hippocampus of the lesion group was not significantly higher than that of the sham group, a clear upward trend was observed. HPA axis activity is negatively regulated by the binding of endogenous glucocorticoids to their receptors.23 The hippocampus has a rich glucocorticoid receptor expression crucial in the feedback regulation of the HPA axis.24 Hb lesions may inhibit the function of the HPA axis by upregulating glucocorticoid receptors in the hippocampus. Collectively, Hb lesions could improve glucose metabolism in T2DM rats by inhibiting the activity of the HPA axis, by enhancing the expression of BDNF and its receptors. Furthermore, a decrease in epinephrine (EP) (p<0.001, figure 5A) and an increase in acetylcholine (p<0.05, figure 5B) in the adrenal glands, as well as a decrease in EP levels in the liver (p<0.001, figure 5C), suggested that Hb lesions could cause peripheral sympathetic nervous system depression (figure 5A). Overall, the inhibition of Hb lesions in the HPA axis and sympathetic nervous system may be essential for mediating the regulation on liver glucose metabolism.
Figure 4Effects of habenular nucleus lesions on the hippocampus of rats. (A) Western blot and statistic results of BDNF in four groups of rats (n=4). (B) Western blot and statistic results of TrkB in four groups of rats (n=4). (C) Western blot and statistic results of the IR in rat hippocampus (n=7). (D) Western blot and statistic results of the GR in rat hippocampus (n=7). *P<0.05. BDNF, brain-derived neurotrophic factor; Ctrl, control; GR, glucocorticoid receptor; IR, insulin receptor; N.S., no significance; T2DM, type 2 diabetes mellitus; TrkB, tropomyosin receptor kinase B.
Figure 5Effects of Hb lesions on the peripheral nervous system of normal rats. (A,B) Concentrations of EP and Ach in adrenal of rats (n=4). (C) Concentration of EP in the liver of rats (n=6). (D) Effects of Hb lesions on serum glucose levels in normal rats, Sham: n=12; lesion: n=9. (E,F): Effects of Hb lesions on the oral glucose tolerance test of normal rats (sham: n=12, lesion: n=9). (G,H) Effects of Hb lesions on the pyruvate tolerance test of normal rats (sham: n=12, lesion: n=9). (I) Dynamic observation of high-carbohydrate and fat diet on serum glucose levels in rats after Hb lesions (sham: n=3, lesion: n=4). *P<0.05, **P<0.01, ***P<0.001. Ach: acetylcholine; AUC, area under curve; EP, epinephrine; FBG, fasting blood glucose; Hb, habenular nucleus; T2DM, type 2 diabetes mellitus.
Studies by Soto et al have shown that IR deletion in the hippocampus and central amygdala result in glucose intolerance in mice.25 In our study, the expression levels of IR in the hippocampus of T2DM rats were lower than those in the control (figure 4C). Compared with the sham group, the expression of IR in the hippocampus of Hb lesion rats was significantly increased (figure 4C), and the rats in the lesion group indicated stronger glucose tolerance in OGTT (figure 2D,E). This evidence suggests that the increased IR expression in the hippocampus may be the main reason mediating Hb lesions to improve glucose tolerance in T2DM rats.
Effects of Hb lesions on blood glucose, glucose tolerance, and hepatic gluconeogenesis in rats under physiological conditions
We observed the effects of Hb lesions on glucose metabolism in diabetic rats, but the role of Hb lesions under physiological conditions is unclear. Hence, we applied the same method and ablated bilateral Hb in normal rats and revealed that the blood glucose levels in the lesions group was also significantly reduced, lower than that of the sham group (p<0.001, figure 5D). Hb lesions decreased the level of blood glucose in normal rats. This result indicates that under normal physiological conditions, Hb has a certain regulatory effect on glucose metabolism in rats. Increased Hb activity results in increased blood glucose production or/and decreased insulin sensitivity. Therefore, we examined the effect of Hb lesions on glucose tolerance in normal rats. OGTT demonstrated that Hb lesions increase insulin sensitivity in normal rats (p<0.05; figure 5E,F), but PTT results indicated that Hb lesions did not affect gluconeogenesis in normal rats (figure 5G,H). Based on the aforementioned two experiments, it can be observed that Hb lesions possess no visible regulatory effect on gluconeogenesis in normal rats but can significantly alter the glucose tolerance of normal rats. Under normal physiological conditions, Hb lesions mainly regulate glucose metabolism by regulating insulin sensitivity.
To determine whether Hb lesions had a preventive effect on diabetes, we first lesioned the Hb 1 week before induction and then induced diabetes using HCFD and STZ. We observed that Hb lesions did not prevent the onset of diabetes, but demonstrated some delaying effects on the rate of rise in blood glucoselevels (figure 5I). These results suggest that Hb lesions can only increase insulin sensitivity in normal rats but have no effect on gluconeogenesis under physiological conditions. Hb lesions do not possess diabetes preventive effects.