Use of reagent for knocking down or inhibiting SLC35f6 in preparation of drug for activating ampk

Abstract

The present application relates to the technical field of biology, and discloses a use of a reagent for knocking down or inhibiting SLC35F6 in preparation of a drug for activating AMPK. The inventor of the present application finds through research that SLC35F6 can be used as a new target for AMPK activity regulation, and finds through further research that an SLC35F6-ALPP complex inhibits the activity of AMPK. According to the present application, it is found that AMPK can be activated by knocking down SLC35F6 or ALPP, and targeting SLC35F6 or ALPP, selecting proper siRNA, and efficiently knocking down SLC35F6 or ALPP provide a new technical solution for the activation of AMPK. According to the present application, the regulation effect of SLC35F6 on the activity of AMPK is clarified for the first time, a new preventive and therapeutic drug research and development approach and drug action target are provided for diagnosis and treatment of diseases related to the activity of AMPK, and the present application has very important medicinal value.

Description

Use of reagents for knocking down or inhibiting SLC35F6 in the preparation of drugs for activating AMPK Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to the use of an agent for knocking down or inhibiting SLC35F6 in the preparation of a drug for activating AMPK. Background Art

[0002] AMPK (Adenosine 5'-monophosphate (AMP)-activated protein kinase), also known as an AMP-dependent protein kinase, is a key regulator of biological energy metabolism. This protein molecule is widely present in animal and plant cells and is central to the study of diabetes and other metabolic diseases. It is expressed in various metabolic organs and can be activated by various stimuli, including cellular stress, exercise, and many hormones and substances that affect cellular metabolism. AMPK acts as a nutrient and energy sensor, sensing intracellular ATP levels. When mitochondria are inhibited and energy supply is low, AMPK phosphorylates specific enzymes and sites, increasing ATP production and reducing ATP consumption, thereby restoring energy balance.

[0003] AMPK is a crucial kinase in metabolic regulation. When intracellular energy is deficient, AMPK is activated by phosphorylation by upstream kinases. This phosphorylation leads to the phosphorylation of numerous key metabolic enzymes and control proteins, promoting catabolism, inhibiting anabolism, and increasing ATP accumulation, thereby providing cellular energy. Traditionally, AMPK activation is believed to result from elevated intracellular AMP concentrations, which bind to the AMPK γ subunit, altering the conformation of the AMPK heterotrimer and enabling phosphorylation of the α subunit at T172 by the upstream kinase LKB1, leading to activation of the AMPK holoenzyme. Recent studies have revealed that AMPK activation is not uniform across subcellular compartments. AMPK can be activated in lysosomes, where its lysosomal localization is dependent on myristoylation of the second glycine in the AMPK β subunit. Furthermore, in addition to responding to AMP, lysosomal AMPK can also respond to changes in the concentration of FBP (fructose-1,6-bisphosphate), a secondary product of glucose metabolism. The proteins on the lysosome that regulate AMPK activity and whether these proteins could serve as potential drug targets for influencing AMPK activity remain unknown.

[0004] The regulatory mechanisms of existing AMPK activators primarily focus on increasing AMPKα phosphorylation, and the regulatory sites involved are already saturated. Currently, there is no mechanism for AMPK dephosphorylation to explore and discover AMPK inhibitors and activators.

[0005] Summary of the Invention

[0006] The purpose of this application is to overcome the deficiencies of the prior art and provide the use of a reagent for knocking down or inhibiting SLC35F6 in the preparation of a drug for activating AMPK.

[0007] To achieve the above-mentioned purpose, the technical solution adopted by this application is: the use of SLC35F6 in the preparation of AMPK activity inhibitors. The purpose of this application is to explore the lysosomal activation mechanism of AMPK and to discover new AMPK activity regulatory proteins. Studies have shown that AMPK on lysosomes can sense changes in secondary products of sugar metabolism, which is different from the regulation of AMPK activity in the entire cell. Therefore, there should be new AMPK regulatory proteins on lysosomes that can more directly regulate AMPK activity. This application uses BiFC and proximity labeling technology to discover that the SLC35F6-ALPP complex inhibits the activity of AMPK on lysosomes, and the SLC35F6 ligand UDP-GlcNAc can relieve the inhibition of the SLC35F6-ALPP complex on AMPK, thereby activating AMPK and exerting therapeutic effects.

[0008] The present application also provides the use of an agent for knocking down or inhibiting SLC35F6 in the preparation of a drug for activating AMPK.

[0009] As a preferred embodiment of the application described in the present application, the reagent includes siRNA that knocks down the expression of the SLC35F6 gene.

[0010] As a preferred embodiment of the application described in the present application, the nucleotide sequence of the siRNA is shown in any one of SEQ ID NO.1 to SEQ ID NO.3.

[0011] The present application also provides the use of an agent for knocking down or inhibiting ALPP in the preparation of a drug for activating AMPK.

[0012] As a preferred embodiment of the application described in the present application, the reagent includes siRNA that knocks down the expression of the ALPP gene.

[0013] As a preferred embodiment of the application described in the present application, the nucleotide sequence of the siRNA is shown in any one of SEQ ID NO.4 to SEQ ID NO.6.

[0014] The present application also provides an AMPK activator, which includes at least one of an agent for knocking down SLC35F6 and an agent for knocking down ALPP.

[0015] The present application also provides the use of at least one of an agent for knocking down SLC35F6 and an agent for knocking down ALPP in the preparation of a product for preventing and / or treating tumors, inflammation or diabetes.

[0016] As a preferred embodiment of the application described in this application, the reagent includes UDP-GlcNAc.

[0017] Beneficial effects of the present application: The present application provides the use of reagents for knocking down or inhibiting SLC35F6 in the preparation of drugs that activate AMPK. The inventors of the present application have found that SLC35F6 can serve as a new target for regulating AMPK activity, and through further research, they found that the SLC35F6-ALPP complex inhibits the activity of AMPK on lysosomes. The present application found that knocking down SLC35F6 or ALPP can activate AMPK. By targeting SLC35F6 or ALPP, appropriate siRNA is selected to efficiently knock down SLC35F6 or ALPP, providing a new technical solution for the activation of AMPK. The present application clarifies for the first time the regulatory effect of SLC35F6 on AMPK activity, provides new preventive and therapeutic drug development pathways and drug targets for the diagnosis and treatment of diseases related to AMPK activity, and has very important medicinal value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a graph showing the expression results of p-AMPK, AMPK, SLC35F6, and tubulin after knocking down SLC35F6 in Example 1;

[0019] Figure 2 is a graph showing the expression results of p-AMPK, AMPK, p-ACC1, ACC1, ALPP, and tubulin after knocking down ALPP in Example 2;

[0020] FIG3 is a graph showing the expression of GST, p-AMPK, AMPK, and tubulin in the cell lysate and beads after immunoprecipitation in Example 3;

[0021] FIG4 is a graph showing the expression results of p-AMPK, AMPK, p-ACC1, ACC1, SLC35F6, and tubulin after SLC35F6 knockdown and starvation treatment in Example 4;

[0022] Figure 5 is a graph showing the expression results of p-AMPK, AMPK, p-ACC1, ACC1, SLC35F6, and tubulin after SLC35F6 knockdown and H2O2 treatment in Example 4;

[0023] FIG6 is a graph showing the expression results of p-AMPK, AMPK, p-ACC1, ACC1, SLC35F6, and tubulin after SLC35F6 knockdown and AICAR treatment in Example 4. DETAILED DESCRIPTION

[0024] The following is a detailed description of the above content of this application through specific implementation methods in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of this application to the following examples. All technologies implemented based on the above content of this application fall within the scope of this application.

[0025] Example 1

[0026] This example experimentally verifies the effect of knocking down SLC35F6 on AMPK activity. The specific experimental steps are as follows:

[0027] (1) HEK293T cells in good growth condition were added to a six-well plate and cultured at 37°C and 5% CO2;

[0028] (2) A control siRNA (provided by Guangzhou Ruibo Biotechnology, catalog number #siN0000001-1-5) with a final concentration of 100 nM and an experimental siRNA for knocking down SLC35F6 at 100 nM were mixed with 10 μl of RNAimax reagent and 200 μl of Optimen to obtain a mixed solution; the sequences of the experimental siRNAs were:

[0029] Experimental group 1 (#1): 5′-GCAUGGUGUUGGACAGCUU-3′ (SEQ ID NO: 1);

[0030] Experimental group 2 (#2): 5′-GCAUUGCCUUCUUCAACUU-3′ (SEQ ID NO: 2);

[0031] Experimental group three (#3): 5′-GAGGAGAAGUUCGUCUACA-3′ (SEQ ID NO: 3).

[0032] (3) The mixture was allowed to stand at room temperature for 20 minutes and then added to HEK293T cells in a six-well plate. Fresh culture medium was replaced after 6 hours. After 48 hours, the cells were collected and lysed, centrifuged at 1000g for 10 minutes, the supernatant was collected, and the protein was extracted.

[0033] (4) Immunoblotting: The protein obtained in step (3) was subjected to polyacrylamide gel electrophoresis with the following electrophoresis parameters: constant voltage 80 V for 20 min plus 120 V for 45 min, then constant current 0.2 A for 1 h for transfer, and blocked with 5% skim milk for 1 h; incubated overnight with primary antibodies against p-AMPK, AMPK, SLC35F6, and tubulin, and then applied with corresponding secondary antibodies the next day to detect the expression levels of the corresponding proteins.

[0034] The experimental results are shown in Figure 1. p-AMPK (T172) indicates the phosphorylation level of AMKP, representing AMPK activity. As shown in Figure 1, the experimental group did not express SLC35F6, indicating successful knockdown of SLC35F6. The control group had lower p-AMPK expression than the experimental group, indicating that AMPK was activated after SLC35F6 knockdown.

[0035] Example 2

[0036] This example experimentally verifies the effect of knocking down ALPP on AMPK activity. The specific experimental steps are as follows:

[0037] (1) HEK293T cells in good growth condition were added to a six-well plate and cultured at 37°C and 5% CO2;

[0038] (2) A control siRNA with a final concentration of 100 nM and an experimental siRNA for knocking down ALPP at 100 nM were mixed with 10 μl of RNAimax reagent and 200 μl of Optimen to obtain a mixed solution; the sequences of the experimental siRNAs were:

[0039] Experimental group 1 (#1): 5′-CUACGCAGCUCAUCUCCAA-3′ (SEQ ID NO: 4);

[0040] Experimental group 2 (#2): 5′-CUGGAGACAUGAAAUACGA-3′ (SEQ ID NO: 5);

[0041] Experimental group three (#3): 5′-CGGUCCUCCUAUACGGAAA-3′ (SEQ ID NO: 6).

[0042] (3) The mixture was allowed to stand at room temperature for 20 minutes and then added to HEK293T cells in a six-well plate. Fresh culture medium was replaced after 6 hours. After 48 hours, the cells were collected and lysed, centrifuged at 1000g for 10 minutes, the supernatant was collected, and the protein was extracted.

[0043] (4) Immunoblotting: The proteins obtained in step (3) were subjected to polyacrylamide gel electrophoresis with the following electrophoresis parameters: constant voltage 80 V for 20 min plus 120 V for 45 min, then constant current 0.2 A for 1 h for transfer, and blocked with 5% skim milk for 1 h; incubated overnight with primary antibodies against p-AMPK, AMPK, p-ACC1, ACC1, ALPP, and tubulin, and then applied with corresponding secondary antibodies the next day to detect the expression levels of the corresponding proteins.

[0044] The experimental results are shown in Figure 2. As shown in Figure 2, the experimental group did not express ALPP, indicating that the experimental group successfully knocked down ALPP; the expression of p-AMPK in the control group was lower than that in the experimental group, indicating that AMPK was activated after knocking down ALPP.

[0045] Example 3

[0046] This example experimentally verifies the effect of reducing the interaction between SLC35F6 and ALPP on AMPK activity. The specific experimental steps are as follows:

[0047] (1) HEK293T cells in good growth condition were added to a six-well plate and cultured at 37°C, 5% CO2; Flag-SLC35F6 and GST-ALPP were transfected into HEK293T cells using PEI, and the medium was changed after 6 hours;

[0048] (2) After 48 h, cells were lysed, and UDP-GlcNAc was added to the cell lysate and incubated for 1 h;

[0049] (3) After 2 h, GST beads were added to the lysate and incubated with rotation for 2 h;

[0050] (4) After beads precipitation, wash three times with lysis buffer;

[0051] (5) Immunoblotting was used to detect the levels of GST, p-AMPK, AMPK, and tubulin in cell line lysates and beads after immunoprecipitation.

[0052] The experimental results are shown in Figure 3. Flag and GST represent overexpressed Flag-SLC35F6 and GST-ALPP. In the 5% input group, it represents the total content, and in the IP group, it represents the components pulled down by immunoprecipitation. The higher the IP / Input ratio, the stronger the mutual binding. As can be seen from Figure 3, compared with the Veh control group (containing only 1X PBS), the addition of UDP-GlcNAc increases p-AMPK, indicating that UDP-GlcNAc can enhance the activity of AMPK (compare the second and third lanes). At the same time, UDP-GlcNAc can reduce Flag-SLC35F6 after GST-ALPP immunoprecipitation by GST magnetic beads, indicating that UDP-GlcNAc can reduce the interaction between GST-ALPP and Flag-SLC35F6. In summary, UDP-GlcNAc can increase the activity of AMPK by reducing the interaction between SLC35F6 and ALPP.

[0053] Example 4

[0054] This example verifies the effect of knocking down SLC35F6 combined with conventional AMPK activators on AMPK activity. The specific experimental steps are as follows:

[0055] (1) HEK293T cells in good growth condition were added to a six-well plate and cultured at 37°C and 5% CO2;

[0056] (2) The control siRNA (final concentration: 100 nM) and the experimental siRNA (final concentration: 100 mM) for knocking down SLC35F6 were mixed with 10 μl of RNAimax reagent and 200 μl of Optimen to obtain a mixed solution;

[0057] (3) The mixture was allowed to stand at room temperature for 20 min, then added to HEK293T cells in a six-well plate, and fresh culture medium was replaced after 6 hours;

[0058] (4) After 48 h, the cells were starved: glucose-free medium was used instead of normal medium for 16 h; hydrogen peroxide treatment: 1 mM H2O2 was added to the normal cultured cells for 5 min; AICAR treatment: AMPK activator 2 mM AICAR was added to the normal cultured cells for 4 h;

[0059] (5) Collect and lyse the cells, centrifuge at 1000 g for 10 min, collect the supernatant, and extract the protein;

[0060] (6) The proteins were detected by immunoblotting: polyacrylamide gel electrophoresis, electrophoresis parameters: constant voltage 80V, 20min plus 120V, 45min, then constant current 0.2A, 1h transfer, and 5% skim milk blocking for 1h; incubated with primary antibodies p-AMPK, AMPK, p-ACC1, ACC1, SLC35F6, tubulin overnight, and then applied with corresponding secondary antibodies the next day to detect the expression levels of the corresponding proteins.

[0061] The results are shown in Figures 4 to 6. As shown in Figures 4 to 6, conventional AMPK activators (such as glucose starvation for 16 hours, hydrogen peroxide treatment for 5 minutes, and AMP analog AICAR treatment for 4 hours) can all increase AMPK phosphorylation (compare lanes 1 and 2). Knockdown of SLC35F6 combined with conventional AMPK activators can further activate already activated AMPK (compare lanes 2 and 4), indicating that knockdown of SLC35F6 combined with conventional AMPK activators can further enhance AMPK activity.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application and are not intended to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. Application of SLC35F6 in the preparation of AMPK activity inhibitors.

2. Use of reagents that knock down or inhibit SLC35F6 in the preparation of drugs that activate AMPK.

3. The use according to claim 2, characterized in that: The reagents include siRNA that knocks down the expression of the SLC35F6 gene.

4. The use according to claim 3, characterized in that: The nucleotide sequence of the siRNA is shown in any one of SEQ ID NO.1 to SEQ ID NO.

3.

5. Use of reagents that knock down or inhibit ALPP in the preparation of drugs that activate AMPK.

6. The use according to claim 5, characterized in that: The reagents include siRNA that knocks down the expression of ALPP gene.

7. The use according to claim 6, characterized in that: The nucleotide sequence of the siRNA is shown in any one of SEQ ID NO.4 to SEQ ID NO.

6.

8. An AMPK activator, characterized in that The activator includes at least one of an agent for knocking down SLC35F6 and an agent for knocking down ALPP.

9. Use of at least one of an agent for knocking down SLC35F6 and an agent for knocking down ALPP in the preparation of a product for preventing and / or treating tumors, inflammation or diabetes.

10. The use according to claim 9, characterized in that: The reagent includes UDP-GlcNAc.

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