KR20230132377A - Composition for preventing or treating obesity, diabetes or NASH comprising SHLP2 - Google Patents

Abstract

The present invention relates to a composition for preventing or treating obesity, diabetes or NASH, containing SHLP2 as an active ingredient. By confirming the strong anti-diabetic, anti-obesity effect and NASH treatment effect of SHLP2 and elucidating the regulatory mechanism, the prevention and treatment of diabetes, obesity, Development as a treatment for fatty liver and NASH is expected. In addition, due to the characteristics of peptide drugs, they exhibit strong activity and diverse actions in vivo, can be used to treat diseases even in relatively small amounts, and have a clinical success rate that is more than twice that of low-molecular-weight compounds, making SHLP2 a powerful agent in the future. The effects of treating diabetes, anti-obesity, and NASH are expected to have a large industrial ripple effect in the development of new drugs.

Description

Composition for preventing or treating obesity, diabetes or NASH comprising SHLP2 as an active ingredient {Composition for preventing or treating obesity, diabetes or NASH comprising SHLP2}

The present invention relates to a composition for preventing or treating obesity, diabetes, or nonalcoholic steatohepatitis (NASH), containing small humanin like peptide 2 (SHLP2) as an active ingredient.

As the number of obese patients has rapidly increased worldwide over the past 20 years, adult diseases such as high blood pressure, hyperlipidemia, fatty liver, and diabetes are also on the rise. Among these, type 2 diabetes is known to be a metabolic disease with the highest risk of occurrence due to overweight and obesity. It is mediated by insulin resistance as a common etiological mechanism, and although genetic factors related to obesity do not correspond to each other, they coexist and are related to each other. It is done. In addition, obesity is closely related to the increase in metabolic syndrome, which is a combination of risk factors for various cardiovascular diseases and type 2 diabetes in one individual. Therefore, the development of treatments and treatments for metabolic syndrome, including obesity and type 2 diabetes, is actively underway both domestically and globally. However, the pathogenesis revealed is still unclear, and the treatments currently being used also affect the central nervous system. Limitations have been reported, such as causing depression and drug dependence by acting on the nervous system and causing serious side effects in the nervous and cardiovascular systems, so the development of new alternative drugs is necessary.

SHLP2 is a peptide encoded in the genome of the mitochondrial 16S ribosomal RNA region and produced in the mitochondria themselves. It is related to peptides derived from mitochondria (Humanin, MOTS-c, Small humanin like peptide 1-6) and is associated with aging, neuropathy, degeneration, It was first discovered in the laboratory of Pinchas Cohen, who is currently conducting a variety of research, including cancer and diabetes. SHLP2 is a peptide consisting of a total of 26 amino acids, and is known to reduce reactive oxygen species (ROS), improve mitochondrial metabolism, and improve defense in vivo. However, little has been revealed about the mechanisms related to this phenomenon, and there has been no research on regulation through the nervous system.

US registered patent US 8,637,470 B2 (registered on January 28, 2014)

Accordingly, the purpose of the present invention is to provide a composition for preventing, improving, or treating obesity, diabetes, or NASH, containing SHLP2 as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating obesity containing SHLP2 as an active ingredient.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating diabetes containing SHLP2 as an active ingredient.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating NASH containing SHLP2 as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving obesity, diabetes, or NASH, containing SHLP2 as an active ingredient.

The present invention relates to a composition for preventing or treating obesity, diabetes or NASH, containing SHLP2 as an active ingredient. By confirming the strong anti-diabetic, anti-obesity effect and NASH treatment effect of SHLP2 and elucidating the regulatory mechanism, the prevention and treatment of diabetes, obesity, Development as a treatment for fatty liver and NASH is expected. In addition, due to the characteristics of peptide drugs, they exhibit strong activity and diverse actions in vivo, can be used to treat diseases even in relatively small amounts, and have a clinical success rate that is more than twice that of low-molecular-weight compounds, making SHLP2 a powerful agent in the future. The effects of treating diabetes, anti-obesity, and NASH are expected to have a large industrial ripple effect in the development of new drugs.

Figure 1 shows the results of SHLP2 antibody development and validation for SHLP2 detection.
Figure 2 shows the results of monitoring changes in SHLP2 according to metabolic disease through serum survey of normal people and metabolic disease patients.
Figure 3 shows the results of feeding and weight loss effects through peripheral regulation of SHLP2.
Figure 4 shows the results of the glucose homeostasis regulation and improvement effect of SHLP2.
Figure 5 shows the results of the effect of SHLP2 on increasing energy consumption.
Figure 6 shows the results of the effect of SHLP2 on regulating thermogenic gene expression.
Figure 7 shows the results of investigation of feeding neurons activated by SHLP2.
Figure 8 shows the results of the feeding and weight reduction effects of SHLP2 through central delivery.
Figure 9 shows the results of thermogenic gene changes caused by ICV SHLP2 injection.
Figure 10 shows the effect of SHLP2 on controlling glucose homeostasis and improving metabolic diseases through central delivery.
Figure 11 shows the effect of irradiating feeding neurons activated by Central SHLP2.
Figure 12 shows the regulatory effect of the hypothalamic melanocortin system by SHLP2.
Figure 13 shows the regulatory effect of the hypothalamic melanocortin system by SHLP2.
Figure 14 shows the development of a fatty liver animal model through Methionine & Choline Deficient dietary induction and the treatment and inhibitory effect of non-alcoholic fatty liver disease by SHLP2.
Figure 15 shows the effect of inhibiting fat accumulation and improving fatty liver by SHLP2 in the liver of a fatty liver animal model.
Figure 16 shows the effects of SHLP2 on inhibiting collagen accumulation and improving liver fibrosis in the liver of a fatty liver animal model.

The present invention provides a pharmaceutical composition for preventing or treating obesity containing SHLP2 as an active ingredient.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating diabetes containing SHLP2 as an active ingredient.

Preferably, the pharmaceutical composition can increase O ₂ consumption, CO ₂ production and heat generation, but is not limited thereto.

Preferably, the pharmaceutical composition can increase the expression of UCP1, PGC1α, Dio2, PRDM16 and NRF1, but is not limited thereto.

Preferably, the pharmaceutical composition is capable of activating hypothalamic feeding neurons, and more preferably, the hypothalamic feeding neurons may be pro-opiomelanocortin (POMC) neurons in the arcuate nucleus (ARC) region. It is not limited to this.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating NASH containing SHLP2 as an active ingredient.

Preferably, the pharmaceutical composition can inhibit collagen accumulation in the liver and improve liver fibrosis, but is not limited thereto.

The pharmaceutical composition of the present invention can be prepared using pharmaceutically suitable and physiologically acceptable auxiliaries in addition to the active ingredients, and the auxiliaries include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, and glidants. Alternatively, solubilizers such as flavoring agents may be used. For administration, the pharmaceutical composition of the present invention can be preferably formulated as a pharmaceutical composition containing one or more pharmaceutically acceptable carriers in addition to the active ingredient. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these ingredients can be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

The pharmaceutical preparation form of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions, and sustained-release preparations of the active compound. You can. The pharmaceutical composition of the present invention can be administered in any conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. It can be administered. The effective amount of the active ingredient of the pharmaceutical composition of the present invention refers to the amount required for the prevention or treatment of disease. Therefore, the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of dosage form and the patient's age, weight, general health condition, gender and diet, administration time, administration route and composition. It can be adjusted depending on a variety of factors, including secretion rate, duration of treatment, and concurrent medications.

In addition, the present invention provides a health functional food composition for preventing or improving obesity, diabetes, or NASH, containing SHLP2 as an active ingredient.

The health functional food composition of the present invention may be provided in the form of powder, granules, tablets, capsules, syrup, or beverage. The health functional food composition is used with other foods or food additives in addition to the active ingredients, and is administered according to a conventional method. It can be used appropriately. The mixing amount of the active ingredient can be appropriately determined depending on its purpose of use, for example, prevention, health, or therapeutic treatment.

The effective dose of the active ingredient contained in the health functional food composition can be used in accordance with the effective dose of the pharmaceutical composition, but in the case of long-term intake for the purpose of health and hygiene or health control, it should be less than the above range. It is certain that the active ingredient can be used in amounts exceeding the above range because there is no problem in terms of safety.

There are no particular restrictions on the types of health foods, and examples include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, Examples include drinks, alcoholic beverages, and vitamin complexes.

"SHLP2" used in the present invention is Small humanin like peptide 2, which contains a total of 26 amino acids (Met-Gly-Val-Lys-Phe-Phe-Thr-Leu-Ser-Thr-Arg-Phe-Phe-Pro-Ser It is a peptide consisting of -Val-Gln-Arg-Ala-Val-Pro-Leu-Trp-Thr-Asn-Ser; SEQ ID NO: 1). Meanwhile, “JP” described in the drawings of this specification refers to “SHLP2”.

Hereinafter, the present invention will be described in detail through examples to aid understanding. However, the following examples only illustrate the content of the present invention and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Experimental example>

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

One. peptide synthesis

The SHLP2 peptide was reacted with dimethylformamide (DMF) on Fmoc-Ser(tBu)-Wang resin. The amino acids were then deprotected using a 20% piperidine solution in DMF. Binding was performed in DMF using hexafluorophosphate Benzotriazole Tetramethyl Uronium (HBTU) and N-methylmorpholine (NMM). Step-by-step deprotection and conjugation of amino acids were repeated until the SHLP2 peptide was synthesized. Peptides were isolated from dried resin using a trifluoroacetic acid (TFA) solution containing 2.5% 1,2 ethanedithiol (EDT), 5% thioanisole, and 5% distilled water. It has been done. The peptide was precipitated in ether and dried under vacuum. Peptides were purified using a column (YMC-Triart C18/S-5 μm/12nm. 5μm (20 x 250 mm)) and each fraction was obtained at a flow rate of 1 mL/min. The molecular weight of the collected fractions was measured using a molecular weight analyzer (HPLC; Shimadzu HPLC LabSolution and MALDI-TOF MS; AXIMA Assurance, Shimadzu). Fractions containing pure peptides were mixed and freeze-dried.

2. Antibody production

1 mL of SHLP2 peptide was injected subcutaneously into two New Zealand white rabbits (0.8-2.0 kg). Rabbits were then boosted twice with the same SHLP2 peptide on days 14 and 28, and mice were sacrificed on day 35 for antiserum collection. To sacrifice the mouse, general anesthesia was induced by intraperitoneal injection of Zoletil (0.5mg/10g), and blood was collected by cardiac puncture.

3. western Blot

Cell lysates were prepared in RIPA buffer. The same amount of protein was used for SDS-PAGE electrophoresis, and the separated protein from the gel was transferred to a PVDF membrane, blocked in 5% non-fat milk for 1 hour, and then incubated with the primary antibody at 4°C for 18 hours. Membranes were washed with 1X TBST and then incubated with appropriate HRP-conjugated secondary antibodies. Blots were visualized with ImageQuant™LAS 4000 or X-ray film.

4. Cell culture and transfection

HEK293T cells (ATCC) were cultured in DMEM (Capricorn Scientific) supplemented with 10% fetal bovine serum (FBS; Corning), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). Cells were maintained at 37°C and 5% CO ₂ temperature conditions. For transient transfection, the cells were washed with DMEM medium without FBS, and the control empty vector and the SHLP2 gene-inserted vector were injected into HEK293T cells using lipofectamine (invitrogen).

5. Immunity points Blot analyze

Immunoblot analysis was performed using serum from normal people or people with metabolic diseases. A 0.2μm nitrocellulose membrane was activated with water (Invitrogen, 10977015), and 3μL of the sample was spotted on the nitrocellulose membrane and dried at room temperature for 30 minutes. The membrane was blocked in 5% BSA prepared in 1X Tris Buffered Control Saline-0.1% Tween 20 (TBST) for 1 hour and then incubated with anti-SHLP2 overnight at 4°C. The membrane was washed three times with 1X TBST and then incubated with anti-rabbit IgG (Invitrogen, 31460). Membranes were washed and visualized using ImageQuant™LAS 4000 (GE Healthcare Life Science). Blots were measured using ImageJ software (NIH Image).

6. Quantitative PCR (q- PCR )

RNA was extracted from tissues using TRizol (Thermo Scientific), and equal amounts of RNA were reverse transcribed into cDNA using ReverTra Ace qPCR RT Master Mix containing gDNA remover (Toyobo, FSQ-301). Afterwards, the cDNA template was amplified and quantified on a BioRad CFX Real-Time PCR System (BioRad) using iQ™SYBR® Green Supermix (BioRad, 1708880).

7. Human Subjects

Ethical approval for human peripheral blood sample collection was obtained from the Yonsei University College of Medicine Committee (4-2017-1168)43 and performed in accordance with the Declaration of Helsinki. The human subjects used in the study were selected from 5 normal group, 5 obese group, and 5 type 2 diabetic group. Additionally, peripheral blood samples were collected from fasting and non-fasting human subjects within the normal group.

8. Laboratory animals

Male mice were used in all experiments. Mice were housed in individual cages at 22 ± 1°C with a 12:12 h light/dark cycle (lights automatic on/off from 08:00 to 20:00). Mice were fed a regular diet (LabDiet, 5053) or a high-fat diet (Research Diets, D12492), and water was provided ad libitum. C57BL/6J (Orient Bio), POMC-Cre (JAX No. 005965)21, and R26-tdTomato (JAX No. 007914)22 mice were used in the experiment. POMC-Cre mice were crossed with R26-tdTomato mice to generate POMC-Cre/R26-tdTomato (tdTomatoPOMC-Cre) mice.

9. SHLP2 administration

For IP injection, SHLP2 peptide and control saline were administered at 2 mg/kg daily. Three weeks after injection, body composition was analyzed and metabolic evaluation was performed using the DEXA (Dual-energy X-ray absorptiometry) system (InAlyzer™, MEDIKORS). For ICV injection, mice were handled daily for 3 days to minimize stress response, and after an overnight fast (18 hours), 3 μg of SHLP2 peptide or control saline was administered, and metabolic evaluation was performed 24 hours after injection.

10. Metabolic Phenotype

A metabolic cage study was conducted by assessing indirect calorimetry, food and water consumption, oxygen consumption (VO2), carbon dioxide production (VCO2), and local locomotor activity using the 12-channel PhenoMaster Home Cage System (TSE Systems). After acclimation for 48 hours, physiological parameters were recorded during a 96-hour recording period. Average metabolic parameters from three 24-hour collection cycles were calculated and statistically analyzed.

11. Glucose and insulin tolerance test

Glucose tolerance (GTT) and insulin tolerance (ITT) tests were performed in mice 3 weeks after intraperitoneal (IP) injection or 4 hours after intracerebroventricular (ICV) injection of SHLP2 peptide. For GTT, mice were fasted overnight. ITT was performed in a fed state. D-glucose (1 g/kg body weight, Sigma-Aldrich, G8270) or insulin (1.2 U/kg body weight, Humulin R, Eli Lilly) was administered by IP injection. Blood glucose levels were measured in venous blood using a portable glucose monitor (Contour TS, Ascensia Diabetes Care).

12. Cell patch clamp

POMC-expressing neurons in the ARC were identified by exposure to epifluorescence under an upright microscope (Nikon FN1). The pipette solution contained (in mM) 127 K-methanesulfonat, 5 KCl, 10 HEPES, 0.3 EGTA, 4 Mg-ATP, 0.3 Na-GTP and adjusted to pH 7.3 with KOH and osmolarity of 288 mOsm/kg. adjusted to Patch pipettes have a resistance of 4-6 MΩ when filled with pipette solution. The whole cell configuration was clamped with a brief negative pressure at a holding voltage of -50 mV. Afterwards, the basal resting membrane potential and firing rate were recorded for about 3 minutes, and then SHLP2 was treated for 3 to 5 minutes. Neurons were classified as responsive if there was a change in resting membrane potential of at least 2 mV during the drug treatment period. Access resistance (Ra) was observed throughout the experiment, and cells with Ra greater than 35 MΩ were excluded from the analysis.

13. Histological analysis

The mouse brain was removed, post-fixed in 10% neutral buffered formalin overnight, and dehydrated in 20% sucrose solution until the tissue settled to the bottom of the container. Mouse brains were cryosectioned into 25 μm slices on a LEICA sectioning machine (Leica Biosystems, SM2010R) and stored in antifreeze before use. Brain slices were washed and then incubated in 1X PBS containing 0.25% (v/v) Triton X-100 (PBT) for 30 minutes. Brain sections were blocked in 3% goat serum prepared in 0.1% PBT for 1 hour at room temperature (RT) and then incubated with c-Fos antibody (Cell Signaling, 2250) overnight at 4°C. Washed brain sections were incubated in anti-rabbit secondary antibodies with Alexa Fluor 488 (Invitrogen, A21206) or Alexa Fluor 594 (Invitrogen, A11012) prepared in PBT containing 3% goat serum. Sections were washed and mounted on glass slides. Brain slides were visualized with a LEICA fluorescence microscope (LEICA Corporation) or a confocal laser microscope (LSM700).

For UCP1 staining, paraffin sections of formalin-fixed iBAT samples were mounted on glass slides and incubated with UCP1 primary antibody (Abcam, ab10983) in a humidifying chamber overnight at 4°C. Slides were washed five times with 1 (vector, PK-6200). After two washes, the slides were incubated with 3,3'-diaminobenzidine (DAB)-peroxidase substrate solution (0.04% DAB and 0.01% DAB) in PBS for less than 2 minutes. H2O2) and visualized on a Nikon Digital Camera DXM1200 microscope system (Nikon Corporation).

< Example 1> SHLP2 for detection SHLP2 Antibody development and validation

As a result of analyzing SHLP2 and biotin-conjugated SHLP2 peptides in a concentration-dependent manner by applying them to Western blot after SDS-PAGE, it was confirmed that the SHLP2 antibody specifically detected SHLP2 peptide as a single band. In contrast, biotin antibodies were detected only on biotin-binding SHLP2 peptide (Figure 1A). In addition, as a result of analysis in HEK293T cells using the SHLP2 peptide overexpression system (transfection), SHLP2 antibody was specifically detected only in the case of SHLP2 peptide overexpression. However, it was not detected in the empty vector in which the SHLP2 gene was not inserted (Figure 1B). Consistent with this, the amount of SHLP2 peptide decreased by increasing the concentration of the peptide competitor (SHLP2 antibody) was confirmed through peptide competition assay (PCA) (Figure 1C). Overall, the specificity of the SHLP2 antibody for the SHLP2 peptide was confirmed (Figures 1A-C).

< Example 2> Monitor changes in SHLP2 according to metabolic disease through serum survey of normal and metabolic disease patients

To investigate whether serum SHLP2 peptide levels can be changed by metabolic disease states in humans, changes in blood SHLP2 peptides in normal/obese/diabetic groups were observed using immuno-dot blot analysis. At this time, SHLP2 peptide was confirmed to have a tendency to decrease in the serum of the metabolic disease group (obesity, diabetes) compared to the normal group, and was especially noticeable in the diabetes group (Figure 2). As a result, this SHLP2 peptide can be used as an important indicator for treating or preventing metabolic diseases.

< Example 3> SHLP2's through peripheral control feeding and weight loss effect

To confirm the therapeutic effect of SHLP2 in metabolic diseases, the effect of reducing body weight and diet over time was confirmed through intraperitoneal injection for 18 days including a high-fat diet (HFD) to induce obesity. As a result, it was confirmed that weight gain due to diet was suppressed under high-fat diet conditions in the SHLP2 peptide-treated group compared to the control saline solution (Figure 3A-B). A decrease in fat in the treatment group was also confirmed in the images observed through dual-energy radiation absorptiometry (DEXA) (Figure 3C-D). Additionally, histological analysis of white adipose tissue (WAT) further demonstrated a marked reduction in the size of WAT lipid cells in SHLP2 peptide-injected mice (Figure 3E-F). As a result, it was demonstrated that administration of peripheral SHLP2 peptide protected mice from HFD-induced obesity and metabolic disorders.

< Example 4> SHLP2's Glucose homeostasis control and improvement effect

The glucose homeostasis regulation and improvement effect of SHLP2 was confirmed through intraperitoneal injection for 18 days. As a result, it was confirmed that blood sugar was reduced in the SHLP2 peptide-treated group in a non-fasting state compared to the control saline group. Additionally, as a result of evaluating glucose tolerance and insulin resistance, it was confirmed that peripheral injection of SHLP2 peptide improved glucose tolerance and insulin sensitivity (Figure 4B-D). As a result, the possibility of SHLP2 peptide to regulate glucose homeostasis was confirmed .

< Example 5> SHLP2's increased energy consumption and heat generation Gene expression regulation effect

To understand the mechanism of the metabolic effect of SHLP2, metabolic changes were evaluated using a metabolic cage 4 weeks after systemic SHLP2 peptide administration. Systemic SHLP2 peptide administration strongly increased O2 consumption (VO2), CO2 production (VCO2), and thermogenesis without changes in physical activity (Figure 5A-D). Since brown fat (interscapular brown adipose tissue; iBAT) is an organ well known for heat generation mainly through UCP1 (uncoupling protein 1), the thermogenic effect of SHLP2 was further confirmed in iBAT. SHLP2 peptide significantly upregulated Ucp1 as well as genes involved in iBAT thermogenesis, including Pgc1α, Dio2, Prdm16, and Nrf1 (Figure 6A-C). Additionally, iBAT from SHLP2 peptide-injected mice had smaller adipocytes and increased UCP1 protein levels compared to control saline-injected mice (Figure 6B-C).

< Example 6> SHLP2 activated by feeding Neuron investigation

c-Fos immunoreactivity was examined throughout the brain after systemic injection of SHLP2. The highest c-Fos activation was observed in the hypothalamus, a key brain region regulating metabolic homeostasis (Figure 7A-B). The ARC and DMH of the hypothalamus showed a marked increase in c-Fos in the SHLP2 injected group. However, the hypothalamus and the ventromedial nucleus (VMH) of the lateral hypothalamus (LH) did not show any differences between the saline group and the SHLP2 group (Figure 7A-B). As a result, it was confirmed that SHLP2 can directly activate hypothalamic neurons.

< Example 7> Via the nervous system SHLP2's feeding and weight loss effect

In light of the findings, we sought to investigate whether SHLP2 affects whole-body energy homeostasis by acting directly on hypothalamic neural circuits, and observed metabolic changes after direct administration of SHLP2 peptide into the nervous system. A single ICV injection of SHLP2 peptide induced an inhibitory effect on food intake and body weight gain for up to 6 days (Figure 8A).

< Example 8> intracerebroventricular SHLP2 Administration ( ICV )On by heat generation Effect of changing genes, controlling glucose homeostasis, and improving metabolic diseases

ICV administration of SHLP2 peptide consistently increased thermogenesis without changes in locomotor activity even under normal food intake conditions (Figure 8B-D). Thermogenic gene and UCP1 expression was significantly enhanced in iBAT (Figure 9A-B). Additionally, ICV injection of SHLP2 peptide improved glucose tolerance and insulin sensitivity (Figure 10A-C). The results strongly indicate that the thermogenic and anorexigenic effects of SHLP2 peptide are mediated through the central nervous system.

< Example 9> Investigation of feeding neurons activated by central SHLP2 and regulation of hypothalamic melanocortin system

To investigate the neurons that regulate the appetite suppressant and thermogenic effects of SHLP2 peptide, SHLP2 was directly administered to the central region and c-Fos (neuron activity marker) staining was performed. As a result, it was confirmed that after SHLP2 administration, the activity of neurons increased in the hypothalamic region, known as the appetite suppression and thermogenesis control center. Through these results, the feeding-related neurons (ARC, PVH, DMH, LH) targeted by SHLP2 peptide were confirmed, and among them, the activity of ARC and PVH neurons was confirmed to be prominent (Figure 11A-B). Additionally, after SHLP2 administration, c-Fos activation was examined in POMC or AgRP neurons using tdTomatoPOMC-Cre or NPY-GFP mice. ICV injection of SHLP2 peptide primarily induced c-Fos in POMC neurons (41%) rather than NPY neurons (14%) (Figure 12A-C). Next, we performed cellular patch clamp experiments on POMC neurons and further characterized the effect of SHLP2 peptide on POMC neuron activity (Figure 13A). Treatment with SHLP2 peptide (1 μM) depolarized the cells, resulting in changes in the resting membrane potential of responding cells (Figure 13B-D). As a result, it was proven that the feeding neuron targeted by SHLP2 peptide was the POMC neuron in the ARC region, and it was predicted to be a major signaling pathway mediating the metabolic improvement effect of SHLP2.

< Example 10> Development of a fatty liver animal model through Methionine & Choline Deficient dietary induction and treatment and inhibitory effect of non-alcoholic fatty liver disease by SHLP2

8 weeks After developing Methionine & Choline Deficient diet-induced fatty liver disease, we sought to confirm the treatment and inhibitory effects of SHLP2 on non-alcoholic fatty liver disease in a fatty liver animal model through intraperitoneal administration of SHLP2 peptide for 4 weeks. As a result, the color of the liver turned yellow in the CDAHFD group compared to the control saline solution, confirming that fat accumulation was progressing due to CDAHFD induction, and it was confirmed that it was relatively suppressed in the SHLP2 peptide-treated group compared to the control saline-administered group (FIG. 14).

< Example 11> Inhibition of fat accumulation and improvement of fatty liver by SHLP2 in the liver of fatty liver animal model

After feeding Methionine & Choline Deficient diet (CDAHFD) for 8 weeks, SHLP2 peptide was administered for 4 weeks, and as a result of analyzing histopathological changes (H&E staining), the three-dimensional structure of the liver lobule was well maintained in the Control group. On the other hand, in the CDAHFD group, the three-dimensional structure of the liver lobules was lost and a lot of fat was confirmed to be accumulated. However, in SHLP2 administration for 4 weeks, a clear decrease in the size and number of fat particles was confirmed in the SHLP2-treated group compared to the control saline-treated group, and through this, it was confirmed that fatty liver was improved by SHLP2 peptide (FIG. 15).

< Example 12> Inhibition of collagen accumulation and improvement of liver fibrosis by SHLP2 in the liver of fatty liver animal model

We aimed to confirm the effect of SHLP2 on inhibiting collagen accumulation and improving liver fibrosis in the liver of a fatty liver animal model. After supplying Methionine & Choline Deficient diet (CDAHFD) for 8 weeks, SHLP2 peptide was administered for 4 weeks, and histopathological (Masson's trichrome (MT)) changes were analyzed. As a result, compared to the control saline treatment group, the CDAHFD group showed a decrease in liver lobules. Collagen fibers (blue part) were observed to accumulate around the area. However, through administration of control saline or SHLP2 peptide for 4 weeks, it was confirmed that the histological fibrosis changes distributed around the liver lobules of the SHLP2 peptide treated group were weaker than those of the control saline treated group (FIG. 16). Therefore, it was confirmed that liver fibrosis was improved through a decrease in collagen fibers in the SHLP2 treatment group.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

A pharmaceutical composition for preventing or treating obesity containing SHLP2 (Small humanin like peptide 2) as an active ingredient. A pharmaceutical composition for preventing or treating diabetes containing SHLP2 as an active ingredient. 3. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition increases O ₂ consumption, CO ₂ production and heat production. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition increases the expression of UCP1, PGC1α, Dio2, PRDM16, and NRF1. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition activates hypothalamic feeding neurons. The pharmaceutical composition according to claim 5, wherein the hypothalamic feeding neuron is a pro-opiomelanocortin (POMC) neuron in the arcuate nucleus (ARC) region. A pharmaceutical composition for preventing or treating nonalcoholic fatty liver disease (NASH) containing SHLP2 as an active ingredient. The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition inhibits collagen accumulation in the liver and improves liver fibrosis. A health functional food composition for preventing or improving obesity, diabetes, or NASH, containing SHLP2 as an active ingredient.