EA Reduced Body Weight and Systemic Lipid Metabolism, and Improved Blood Glucose Levels
Rats fed with an HFD for 4 weeks tended to become obese. After EA intervention, the body weight of the rats in each group decreased to different degrees (Fig. 1A). Meanwhile, the indexes related to abnormal lipid metabolism, such as triglyceride (TG) and total cholesterol (TCH), increased in the model group. However, these levels recovered after EA, with EA at ST25 having the best effect (Fig. 1B-C). Leptin is a hormone secreted by adipose tissue that is proportional to the size of adipose tissue in animals. The changes in leptin expression suggest that EA might help to correct body adipose accumulation in rats (Fig. 1D). The rats also showed abnormal glucose metabolism, including increased serum glucose levels (Fig. 1E) and a spike in insulin secretion (Fig. 1F). EA was found to have a favorable hypoglycemic effect.
Ea Ameliorated Injury Of Insulin Signaling Cascades In Gastrocnemius Muscle
Abnormal lipolysis of adipocytes is one of the recognized causes of insulin resistance and impaired glucose tolerance [25]. Adipocytes in control group were uniform in size, clear in boundary and closely arranged (Supplementary Fig. 2A). The adipose cells of model group increased in diameter and varied in size (Supplementary Fig. 2B). The number of adipose cells decreased significantly in the same field of vision. Compared with the model group, the treatment group had different degree of improvement (Supplementary Fig. 2C-E).
Moreover, skeletal muscle is a critical site of energy metabolism and is one of the main organs to absorb and utilize glucose and fatty acids. Accordingly, skeletal muscle metabolism disorder is an important part of insulin resistance during T2DM. Abnormal energy metabolism can be seen in obesity. Damage to insulin receptors (InsR), along with reduced insulin binding and utilization, leads to localized accumulation of insulin in the gastrocnemius muscle. In this experiment, the model group developed insulin resistance, along with insulin resistance, which was consistent with the results suggested by serum analysis (Fig. 1G-I). EA could reverse these pathological changes, suggesting that EA at ST25, ST37, and LI11 could regulate systemic energy homeostasis.
Ea Repaired Early Disruption Of Islet Morphology And Pancreatic Function In Obesity
Islet morphology was observed through histological analysis (Fig. 2). In the control group, the islets appeared round (Fig. 2A), which is a typical physiological manifestation. However, the HFD rats showed damage to islet beta cells, such as moderate damage and swelling, as shown in Fig. 2B. After HFD, islet atrophy was observed, with irregular margins and marked vacuoles (blue arrowheads). The islet morphology of each treatment group was repaired to varying degrees (Fig. 2C-E). In the control group, the islet beta cells covered the islets uniformly and the blood supply was abundant. EA reversed those effects. The morphology of ST25 group was similar to that of the control group. The vacuolation of the LI11 and ST37 groups were reduced, and the blood supply was slightly recovered.
Further western blotting analysis verified functional damage of pancreatic beta cells in model rats, as shown in Fig. 2F. Peripheral insulin resistance occurred and islet beta cells secreted increased amounts of insulin, resulting in functional overload. As the carrier of insulin function, InsR parallels the downward trend of insulin, which reflected the progress of insulin resistance (Fig. 2F-G). After EA, overall insulin resistance was improved, the amount of insulin secretion was reduced, and insulin receptors were restored, but the treatment effect of LI11 and ST37 was not significant (P > 0.05, Fig. 2F-H). Peripancreatic astrocytes also contribute to pancreatic homeostasis by regulating the secretion of glial fibrillary acidic protein (GFAP) [26]. The expression of GFAP increased after EA at ST25 or LI11 (P < 0.05, Fig. 2F, H), indicating that EA could repair the islet beta cells and restore their function; however, it showed acupoint specificity.
Effects Of Ea On Regulating Pancreatic Mcrocirculation Blood Perfusion
Pancreatic microcirculation is involved in information transfer and material and energy exchange between tissues and cells, which is the basic environment for the survival of islet cells [27]. The islet is a highly vascularized structure, and pancreatic microcirculation dysfunction inevitably affects pancreatic islet function [28]. In the model group, we observed a significant suppression of pancreatic perfusion (Supplementary Fig. 3A). This suggested that pancreatic microcirculation disorder is the key factor leading to structural changes and functional damage of islet beta cells. Whether in normal group or model group, acupuncture at ST25 improved pancreatic blood flow, which partly explains the recovery effect of EA at ST25 on islet morphology and function (Supplementary Fig. 3B, E). Meanwhile, stimulation at LI11 and ST37 inhibited pancreatic blood microcirculation, which was consistent with poor pancreatic morphological recovery after treatment (Supplementary Fig. 3C&F, D&G). EA at ST25 increased the production of vascular endothelial growth factor (VEGF) in the pancreas, which helps to maintain pancreatic beta cell function [29] (Figure 2F, J).
EA Improved the Remodeling Effect of Adipose Tissue on the PINS
PAT belongs to the white adipose tissue (WAT), and its metabolic activity is controlled by the plasticity of nerve endings [30]. However, whether the blocking of NGF signal transduction leads to the inactivation of adjacent pancreatic nerves in unknown. Results showed that the expression of NGF in the model group was significantly reduced (Fig. 3B, F, G), while that in the intervention group was restored to varying degrees (Fig. 3C-G). This was consistent with the change trend of islet morphology, and the repair and maintenance of islet morphology is related to the abundance of the nervous system [31].
EA Improved Potential Link between the Construction of Adipose and Microcirculation Perfusion
The expansion of adipose tissue is related to its plasticity, which in turn is related to the proliferation of microvessels [32]. Thus, the normal functioning and maintenance of adipose tissue homeostasis are closely related to the operation of the microvascular system. A small amount of fat was present in the normal group, suggesting a nutritional effect of PAT on the pancreas (Fig. 4A). Under abnormal glucose metabolism, adipocyte adaptability increases to facilitate the storage of insulin in the form of vesicles [33]. PAT increases significantly (Fig. 4B), indirectly expanding the cellular space where the microvessels are located. EA treatment could effectively increase PAT blood perfusion in the normal or model group, with an obvious and immediate effect (Supplementary Fig. 3H-N). VEGF diminished in PAT was consistent with that in the pancreas, indicating impaired insulin secretion and decreased beta cell mass (Fig. 3F, H). Furthermore, abnormal levels in VEGF tend to parallel levels of inflammation, indicating further damage [34].
Ea Led To Balanced Inflammatory Factors In Adipose Tissue
The expansion of adipose cells allows them to recruit more macrophages and polarize to a pro-inflammatory state when stimulated by chemokines [35]. Therefore, the occurrence of T2DM will be affected by signal transduction between macrophages and adipocytes, especially peripancreatic adipocytes. As shown Fig. 5, the level of EGF-like module-containing mucin-like hormone receptor-like 1 (also known as F4/80) was elevated in the model group (P < 0.05, Fig. 5A, B), suggesting a large number of macrophages. The secretion of monocyte chemoattractant protein1 (MCP-1) increased sharply (P < 0.05, Fig. 5A, C), the proportion of interleukin (IL)-10 and IL-1β ratio imbalance (P < 0.05, Fig. 5A, D-E), and tissue inflammation was formed. Moreover, IL-1β causes direct damage to islet beta cells [36], which also explains the reason for the impaired islet function in obesity. After EA at ST25 and LI11, the levels of these inflammatory factors were reversed in adipose tissue, as shown in Fig. 5. ST37 had a relatively poor anti-inflammatory effect, showing no obvious regulatory effect on F4/80 (P > 0.05, Fig. 5A, B).
The Weight Loss Effect of EA is Related to the Activation of TRPV1 and the Expression of Genes Related to Lipid Metabolism
Adipocyte fatty acid-binding protein 4 (FABP4) is expressed in mature adipocytes, and peroxisome proliferators-activated receptor γ (PPAR-γ) is a major regulator of adipogenesis [37]. These two indicators can monitor the life course of adipocytes. As a downstream transcription element of PPAR-γ, FABP4 can provide feedback regulation for PPAR-γ [38]. The increased level of FABP4 and the decreased level of PPAR-γ in the model group, verified the disruption of adipocyte differentiation and metabolic homeostasis (Fig. 5F-H). The augment of FABP4 enhanced lipid concentration and restrained insulin sensitivity, while the change of PPAR-γ was negatively correlated with FABP4. EA could restore the expression of them. In addition to being a regulatory factor of lipid texture, they can also regulate the occurrence and development of inflammation and improve insulin sensitivity. Therefore, they are also potential targets for the treatment of T2DM [39]. Consistent with this, we found that the changes in TRPV1 in the model and treatment groups were similar to those of FABP4 and PPAR-γ (Fig. 5F, I). In general, EA at ST25 had the most significant regulatory effect.
Ea At St25 Has Trpv1-dependence On Glucolipid Metabolism Management
In summary, EA at ST25 had stronger lipid-lowering and antiinflammatory effects than LI11 and ST37. Moreover, EA at ST25 could effectively repair islet morphology, supporting the stability of blood glucose, and preventing obesity from progressing to T2DM. Therefore, Trpv1-/- mice were selected to further explore the molecular mechanism of EA at ST25. EA at ST25 could effectively reduce the blood sugar peak and restrain short-term blood sugar fluctuations. The Trpv1-/- mice showed a hypoglycemic effect without treatment (Fig. 6B), suggesting that TRPV1 is a vital molecule to maintain glucose metabolism homeostasis. However, after the Trpv1-/- mice received EA at ST25, the effect was no different from that without treatment, indicating that the effect of EA at ST25 had a strong TRPV1dependence.
To determine whether TRPV1 in the pancreas mediates the above effects, we injected TRPV1 agonists and inhibitors into the pancreatic ducts of wild-type (WT) mice, and observed the reaction to blood glucose of the mice after surgery. As hypothesized, the TRPV1 agonists worsened glycemic fluctuations and the inhibitors contributed to glycemic maintenance after both 2 g/kg and 10 g/kg glucose injections (Fig. 6C, Supplementary Fig. 4C). EA at ST25 also relies on TRPV1 in the pancreas to exert its effect (Fig. 6B, D). Therefore, TRPV1 can not only improve glucose metabolism, but also ensure the effect of ST25. This conclusion was also confirmed by the sharp decrease in the response of PINS to manual acupuncture (MA) after TRPV1 knockout. The activity of PINS during manual acupuncture MA (3.47 ± 0.16 Hz) was significantly increased compared with the pre-MA frequency (0.39 ± 0.02 Hz, P < 0.05, Fig. 7A, B). However, the electrophysiological activity of PINS did not change significantly after acupuncture intervention in the Trpv1-/- mice (0.31 ± 0.02 Hz vs. 0.43 ± 0.07 Hz, P > 0.05, Fig. 7C, D).
Moreover, we also observed changes in wet weight of the PAT and other adipose tissue in the Trpv1-/- mice, indicating that TRPV1 knockout can improve the lipid metabolism in mice, especially the effect on visceral fat such as PAT (Supplementary Fig. 5A). The regulatory effect of TRPV1 on PAT could be reproduced after pancreatic TRPV1 sensory sensitive afferent neurochemical ablation; however, EA at ST25 was not effective at this time (Supplementary Fig. 5B). This suggested that there is neural crosstalk between PAT and islet beta cells, and TRPV1 sensory nerves are one of the carriers, which influenced the effect of EA at ST25. The scant effect of MA at ST25 on the activity of PAT provide support for this hypothesis (Fig. 7G-H). Only the pancreatic TRPV1 afferent nerve was ablated; therefore, the weight loss effect of EA at ST25 on epididymal white adipose tissue (eWAT) remained unchanged (Supplementary Fig. 5B). The expansion of subcutaneous adipose tissue helps maintaining inflammatory response levels, which partly explains why subcutaneous adipose tissue in the Trpv1-/- mice did not change significantly. These changes suggested that TRPV1 also plays a critical role in maintaining lipid metabolism, preventing the progression of insulin resistance by improving signaling in PAT and islet beta cells.
EA at ST25 Restores Endocrine Function by Stimulating the Sensory Afferent TRPV1 and Mediating the TRPV1-CGRP-beta Cell Circuit
Rats fed an HFD showed a series of neuroadaptive changes in the pancreas, one of which was an increase in TRPV1-positive nerves (Supplementary Fig. 6A, D, Fig. 6E). To determine the specific mechanism of TRPV1-mediated pancreatic beta cell and PAT communication, we examined the changes in tissue protein content in Trpv1−/− mice. The knockdown of TRPV1 led to a significant decrease in the level of hyperinsulin in the pancreas, and the degree of inflammation in PAT was also diminished (Fig. 8A-F). Low-grade inflammation of adipose tissue over time is associated with metabolic diseases such as insulin resistance in overweight and obese individuals [40]. Persistent inflammation of PAT will exacerbate the imbalance of energy metabolism. The TRPV1 cation channel plays a vital role in regulating glucolipid metabolism by correcting hyperinsulinemia, improving inflammation of adipose tissue, and balancing the interaction between the pancreas and adipose tissue.
After verifying that sensory afferent TRPV1 is a vital t neurophysiological basis for maintaining the stability of islet function, we explored the cascade molecular mechanism of TRPV1 activation. The reason to focus on the expression of calcitonin gene-related peptide receptor component protein (CGRP) is that it can be regulated by TRPV1, and CGRP has a direct inhibitory effect on insulin [19]. In comparison with the normal control group, the model group showed an increased level of TRPV1 and a decreased level of CGRP. No significant difference was found between the model group and the ST37 and LI11 groups. EA at ST25 restored CGRP expression in the pancreas, whereas LI11 and ST37 did not (Fig. 8G-I). These results are consistent with our observation that EA at ST25 has the best effect and could inhibit insulin secretion by increasing CGRP expression.