Oral administration of bacterial Beta cell expansion factor A (BefA) alleviates diabetes in mice with Type 1 and Type 2 diabetes

Diabetes mellitus (DM) is a group of metabolic diseases, which is of urgent need to develop new therapeutic DM oral drugs with less side effects and sound therapeutic efficacy. In this study, a Beta cell expansion factor A (BefA) production strain of Escherichia Coli BL21-pet 28C-BefA was constructed, and the anti-diabetes effect of BefA was evaluated using type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) mice models. The T1DM mice results indicated that BefA significantly reduced the blood glucose level, exerted protective function of islet β cell morphology, down-regulated the TLR-4, p-NFκB/NFκB, Bax/Bcl-2 expressions and the secretion level of IL-1β, TNF-α, increased the expression of PDX-1 protein and insulin secretion in a concentration-dependent manner, and restored the disturbed microbial diversity to normal level. Similar with the T1DM mice, BefA obviously increased islet β cells, reduced inflammatory reaction and apoptosis in T2DM mice, and also improved liver lipid metabolism by down-regulating the expression of CEBP-α, ACC, Fasn and inhibiting the synthesis of triglyceride and induce Cpt-1, Hmgcs2, Pparα in a concentration-dependent manner. In the present study, we verified therapeutic effect and potential mechanisms of BefA in mammal for the first time, providing basic data for its clinical application.

(T2DM) mice models. The T1DM mice results indicated that BefA significantly 23 reduced the blood glucose level, exerted protective function of islet β cell morphology,   Insulin-dependent type 1 diabetes mellitus (T1DM) and insulin-independent type 2 49 diabetes mellitus (T2DM) are the main types of diabetes, among which T1DM results 50 from the specific deficiency of insulin-producing pancreatic β cell from autoimmune 51 destruction (Jahromi and Eisenbarth, 2007), and T2DM is an age-related disease 52 characterized by the dis-function of glucose metabolism representing insulin-resistant 53 states, accompanied by a destruction of β cell function (Weir and Bonner-Weir, 2013). 54 For the treatment of T1DM, insulin injection therapy was applied after its discovery in 55 1922, and it can only alleviate but fail to eliminate T1DM, and also may cause long-56 term physical suffering through subcutaneous injection (Amiel and Rela, 2005). New 57 methods such as immunotherapy, gene therapy, and organ transplantation have been 58 developed rapidly, but they are still in the research stage due to problems such as but the expensive cost and the need for injection limit its clinical use (Wit et al., 2016). 67 Based on the disadvantages of the above treatment strategies, it is of great importance

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As no work is done to verify the increasing effect of orally administered BefA on the 121 number of mammalian islet β cells, therefore newborn germ-free (GF) mice and specific 122 pathogen free (SPF) mice were used in the present study to confirm the anti-diabetes 123 effect of BefA for the first time, and our results indicated that BefA could markedly 124 increase the β cell number compare with SPF mice and GF mice (Fig. 1C), consistent 125 with previous work in zebrafish (Hill et al., 2016).         concentration-dependent manner (Fig. 2). T1DM will undergo abnormal islet β cell 294 apoptosis, therefore we tested the expressions of Bax/Bcl-2 and PDX-1 in pancreatic 295 tissue. Bax and Bcl-2 are classified as members of Bcl-2 family, among which Bax is 296 up-regulated in apoptosis, and Bcl-2 is an important anti-apoptotic protein, and the ratio 297 Bax/Bcl-2 is often used to evaluate their combined effect (Tomita, 2017). PDX-1, also 298 called insulin promotor 1, is considered as an irreplaceable transcriptional factor in the 299 differentiation and proliferation of islet β cells (Holland et al., 2005). The results 300 indicated that BefA could recover the number of islet β cell and islet function of T1DM 301 mice via reducing the Bax/Bcl-2 ratio, increasing the expression of PDX-1 protein and 302 promoting insulin secretion (Fig. 3).

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More and more studies indicated that intestinal microbiota was closely related to the

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In T2DM mice, BefA also obviously reduced the blood glucose level as well as relieve 315 weight gain symptoms in a concentration-dependent manner (Fig. 4). Notably, the  (Fig. 4, Fig. 5). In addition, the results suggest that BefA can   347 The BefA gene (M001_10165) was codon optimized for Escherichia coli BL21 to 348 favour higher protein yield, and was synthesized with a histidine (His) tag (to facilitate 349 the identification and purification of BefA protein), which was inserted into the

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Another 15 wild-type C57BL/6 mice were used as normal control group (C group).

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Within each group, all mice were used to test blood glucose level (once a week), body 391 weight (once a week) and fecal microbiota structure (at week 7), in which 4 mice were 392 sacrificed for pancreas Western-blotting analysis, 4 mice were sacrificed for pancreas 393 qPCR analysis, the 3 mice were sacrificed for pancreas HE staining, 394 immunehistochemical staining and immunofluorescent staining.   Table 1). Statistical significance was determined using one-way or two-way ANOVA and were 486 annotated using the international convention related to the statistical representation.

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The authors declare no conflict of interest.

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Data availability 490 The authors confirm that the data supporting the findings of this study are available   Regulating liver lipid metabolism TLR-4 p-NFκB IL-1β TNF-α CEBP-α liver lipid accumulation