Bauhinia forficata link extract attenuates insulin resistance by preserving glucose uptake in gastrocnemius muscle

Abstract Bioactive metabolites from Bauhinia forficata Link (Bf extract) hold therapeutic potential for type 2 diabetes mellitus (T2DM) but the mechanism remains poorly understood. This study aimed to test the extract from Bf leaves obtained by decoction on the prevention of T2DM in vivo. The Bf extract was tested on a streptozotocin-induced T2DM mouse model fed on a high-fat diet. The insulin resistance was attenuated in T2DM animals supplemented with Bf extract, which indicates glucose intolerance reduction and p-AKT/AKT ratio preservation in the gastrocnemius muscle. These observations suggested that Bf extract enhanced glucose uptake. Nevertheless, there was no preservation in β-cell insulin secretion in Bf extract-treated T2DM mice. Interestingly, the Bf extract reduced body weight gain without affecting total energy intake. Hence, Bf extract has a hypoglycemic effect which could attenuate the development of insulin resistance. Graphical Abstract


Introduction
Bauhinia forficata Link (Bf) (Fabaceae) is a Brazilian plant known as "cow's paw", that has been shown to possess anti-diabetic properties (Pepato et al. 2002;Lino et al. 2004;Franco et al. 2020;Tonelli et al. 2022). The chemical constituent for the Bf extract is the glycoside flavonoid kaempferol-3,7-O-(a)-dirhamnoside (Kaempferitrin), which is enriched in the leaves (Silva et al. 2000;Cechinel-Filho 2009). More importantly, the Kaempferitrin displays hypoglycemic activity in diabetes-induced rats, performing a beneficial effect on the treatment of type 2 diabetes (T2DM) (Jorge et al. 2004;Sousa et al. 2004;Cazarolli et al. 2013). However, the molecular mechanisms underlying Bf extract activity in insulin-responsive tissues remain unknown, as well as its ultimate potential for preventing the onset of T2DM. Thus, we hypothesized that Bf also could prevent T2DM for maintaining insulin sensitivity and glucose uptake.

Results and discussion
The high-performance liquid chromatography with diode array detector (HPLC-DAD) found five highly abundant compounds in the Bf extract, with different retention times (RT) and polarity (Figure S1A, supplementary material). As shown in the representative chromatogram ( Figure S1B and S1C), the flavonoid Kaempferitrin was detected at 17.72 min (Peak 5). Peak 5 ( Figure S1D) revealed Kaempferitrin by comparing the ultraviolet spectrum ( Figure S1E) to a known standard spectrum ( Figure S1C). The power of Bf against insulin resistance is associated with its composition of polyphenolic compounds (Cechinel-Filho 2009). The type of Bf extract used in this study was identified as a source of polyphenols (Table S2, supplementary material), including the Kaempferitrin mentioned above. This compound could, at least partially, contribute to the improvement in insulin sensitivity described below.
The area under the curve of oral Glucose Tolerance Test (oGTT AUC) was significantly greater for the high-fat diet and streptozotocin (HS) and high-fat diet and streptozotocin (STZ) supplemented with Bf by oral gavage (HSBf) mice when compared to control with normal fat diet group supplemented with water (C) and control supplemented with Bf by oral gavage (CBf) (p < 0.001) ( Figure S2A and S2B, supplementary material). However, the HSBf mice displayed an intermediate response than HS during the test, and there was a trend toward the prevention of glucose intolerance ( Figure  S2B, supplementary material; p ¼ 0.099). HS animals displayed insulin resistance ( Figure  S2C and S2D, supplementary material; p < 0.001) and Bf extract significantly increased the insulin sensitivity when comparing HSBf to HS ( Figure S2D; p ¼ 0.0021). Furthermore, HSBf presented significantly lower fasting glycemia when compared with the diabetic HS group ( Figure S2E; p < 0.001), although there was no difference in fasting insulinemia between these groups ( Figure S2F). The HS also displayed a tendency (p ¼ 0.063) toward reduced functional b-cell capacity when compared to C, and Bf extract was not able to reverse this effect ( Figure S2G). It is important to mention that the hypoglycemic effect was not observed in the CBf group ( Figure S2E). Our results demonstrated that the HS developed a mild-to-moderate T2DM, which was expected with a combination of a high-fat diet (HFD) and low-dose streptozotocin (STZ) injections (Mu et al. 2006;Gilbert et al. 2011). However, Bf attenuated the development of T2DM in HSBf by maintaining glycemic homeostasis at intermediate levels by improved insulin sensitivity and attenuated glucose intolerance.
The islet of Langerhans mean area did not differ between HS and Bf groups (Fig.  S3A), despite the HS mice presenting a reduced functional b-cell capacity. One possible explanation for this fact is that hyperglycemia impairs insulin secretion before apoptosis and that this latter condition is particularly relevant to reduced b cell mass (Donath and Halban 2004). Moreover, Zheng et al. (2015) demonstrated that polyphenols protect islet morphology. Concerning morphology, it is evident that HS animals presented an impaired islet shape and Bf preserved this aspect ( Fig. S3D and S3E, respectively) in comparison to C and CBf animals ( Figure S3B and S3C, respectively).
The gastrocnemius muscle is an insulin target that contributes to the glucose homeostasis. The gastrocnemius of the animal from the HS group had a reduced ratio (p ¼ 0.0314) between phosphorylated and total protein kinase B (p-AKT/AKT) levels in comparison to the control ( Figure S4A and S4B). In contrast, the gastrocnemius of the HSBf group had significantly increased AKT phosphorylation (p-AKT) levels, when compared to the HS group (p ¼ 0.0310). Therefore, Bf was able to maintain the p-AKT/AKT ratio at physiological levels. On the other hand, Bf did not modify the p-AKT levels in the liver or RET (retroperitoneal) fat depot. The AKT is a key protein related to glucose transporter type 4 (GLUT-4) translocation to the membrane and glucose uptake. This result could be particularly relevant since this muscle is the major peripheral tissue responsible for glucose uptake (DeFronzo 2004). Studies have shown that the metabolic pathway of Kaempferitrin on glucose uptake involves the insulin receptor and AKT phosphorylations, and translocation of GLUT-4 in a skeletal muscle (soleus) (Cazarolli et al. 2013) and in 3T3-L1 adipocytes (Tzeng et al. 2009). Indeed, Kaempferitrin functioned as an insulin-mimetic factor, since it increased 14 C-glucose uptake in the soleus muscle of rats (Jorge et al. 2004). Thus, it is possible to hypothesize that the Kaempferitrin, found in our Bf extract, could act solely or synergistically, in the maintenance of glycemic metabolism during partial T2DM prevention. AMPK (5 0 adenosine monophosphate-activated protein kinase) and ERK (extracellular signal-regulated kinases) are associated with glycemic homeostasis and several cellular processes (Roskoski 2012;Zheng et al. 2015;Biondo et al. 2020;Li et al. 2022). However, the hypoglycemic effect of Bf was not associated with the AMPK or ERK pathway ( Figure S4A and S4B). It is probable that the Bf antidiabetic effect only relates to AKT, among the studied pathways.
In this study, the T2DM model did not induce fluctuations in the ratio between c-Jun N-terminal kinase forms (p-JNK/JNK) ( Figure S5). A combination of HFD and doses of STZ demonstrated an increase in total JNK in the liver (Ma et al. 2017). Thus, the lack of any observable alterations in the JNK state suggests that this T2DM-induced approach was not severe enough to cause inflammation, which further supports the assessment of this model as a mild to an intermediate form of T2DM. Besides, Bf administration had not influenced the inflammatory status of these mice.
The prophylactic administration of Bf extract was able to significantly reduce body weight (BW) gain of both CBf and HSBf groups when compared to C (p ¼ 0.0181), and further reduced the BW gain of HSBf animals, when compared to C (p < 0.001) and CBf (p ¼ 0.0026) ( Figure S6A and S6B). These observations in BW happened without any change in the total energy intake, irrespectively of the group ( Figure S6C). Furthermore, energy efficiency was reduced in HSBf mice when compared to C ( Figure  S6D; p ¼ 0.0210). Decreased energy efficiency in HSBf is due to a low ability to convert food intake into body weight. Likewise, polyphenol-rich extracts have been associated with BW loss and energy expenditure increase by stimulating fat oxidation and thermogenesis (Cunha et al. 2013).
Insulin resistance was also associated with steady blood lipid profiles (Table S4) in a similar T2DM mouse model (Gilbert et al. 2011). However, glutamate pyruvate transaminase (GTP) activity was significantly reduced in CBf and HSBf groups, in comparison to the controls, thus indicating the similar hepatoprotective effect to Bauhinia variegata (Bodakhe and Ram 2007). Finally, Bf was not toxic to the kidneys demonstrated by unaltered serum creatinine levels (Pozzobon et al. 2012).

Experimental
The experimental section was supplied as supplementary material.

Conclusion
This study demonstrated that Bf extract partially prevents the HFD and STZ-induced T2DM, as evidenced by enhanced insulin sensitivity, maintained intermediate glycemic levels, a tendency to attenuate glucose intolerance, and stimulated glucose uptake in the gastrocnemius muscle. Additionally, Bf extract did not cause hypoglycemic states in normoglycemic animals. Moreover, the AKT pathway seems to mediate the cellular effects of Bf extract on T2DM-associated alterations.

Disclosure statement
No conflict of interest was reported by the authors.