Nidogen-2 derived peptides for treatment of diabetes

Abstract

Among the various aspects of the present disclosure is the provision of methods for the treatment of type 1 diabetes by administering a brown adipose tissue (BAT)-secreted factor.

Description

[0001] NIDOGEN-2 DERIVED PEPTIDES FOR TREATMENT OF DIABETES

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under DK123301 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0004] FIELD OF THE DISCLOSURE

[0005] The present disclosure generally relates to methods for the treatment of diabetes.

[0006] BACKGROUND

[0007] Type 1 diabetes (T1 D) is defined by autoimmune-mediated destruction of the insulin-producing pancreatic [3-cells and is exacerbated by the aberrant hypersecretion of glucagon by a-cells (Cheon, Korean J Pediatr, 2018, 61 , pp 307- 314). Current therapeutic strategies for T1 D primarily focus on direct or indirect insulin supplementation or whole pancreas / pancreatic islet transplantation (Boscari et al., Rev Endocr Metab Disord, 2021 , 22, pp 217-240). While these methods are effective, the necessity for meticulous insulin dosing and transplant rejection presents challenges to effective T1 D management. Consequently, there is a continuing need to discover and validate additional tools for T1 D therapy (Maffi et al., Rev Diabet Stud RDS, 2011 , 8, pp 44-50 and Jacqueminet et al., Diabetes Metab, 2005, 31 , pp 4S45-44S50).

[0008] SUMMARY

[0009] Among the various aspects of the present disclosure is the provision of methods for the treatment of type 1 diabetes.

[0010] In one aspect, a method of treating type 1 diabetes (T1 D) by administering a therapeutically effective amount of a brown adipose tissue (BAT)-secreted factor is disclosed. In some aspects, the BAT-secreted factor may be a protein or protein fragment from a BAT-conditioned buffer exceeding a molecular weight of 100kDa. In some aspects, the BAT-secreted factor may be a protein or protein fragment from a BAT-conditioned buffer with a molecular weight between 100kDa and 3kDa. In some aspects, the BAT-secreted factor may be a protein or protein fragment from a BAT-conditioned buffer with a molecular weight between 3kDa and 1 kDa. In some aspects, the BAT-secreted factor may be a protein or protein fragment from a BAT-conditioned buffer with a molecular weight smaller than 1 kDa. In some aspects, the BAT-secreted factor may be a protein or protein fragment from a BAT- conditioned buffer that is between 100 and 1 ,000 amino acids, such as between 100 and 800, 100 and 500, 100 and 400, 100 and 300, 200 and 1 ,000, 200 and 800, 200 and 500, 200 and 400, 250 and 500, 300 and 1 ,000, 300 and 800, 300 and 500, 300 and 400, 400 and 1 ,000, 400 and 800, and 400 and 500 amino acids, particularly 250 and 500 amino acids. In some aspects, the protein or protein fragment may be nidogen-2 or a nidogen-2 fragment. In some aspects, the BAT-secreted factor may comprise a synthetic protein or protein fragment. In some aspects, the BAT-secreted factor may be a synthetically modified protein or protein fragment from a BAT-conditioned buffer. In some aspects, the BAT-secreted factor may be a synthetically modified protein or protein fragment from a BAT- conditioned buffer exceeding a molecular weight of 100kDa. In some aspects, the administration of the protein or protein fragment treats T1 D, such as by improving glucose tolerance, improving insulin sensitivity, and / or promoting adipocyte differentiation.

[0011] In another aspect, a method of isolating a BAT-secreted factor comprising incubating BAT cells in contact with media for a sufficient amount of time for BAT- secreted factors to be present and filtering the media by membrane filtration to collect a molecular weight-specific fraction is disclosed. In some aspects, the method further comprises running the molecular weight-specific fraction through an anion exchange chromatography column, running the anion exchange chromatography column fraction through a size exclusion chromatography column, and isolating an active protein fraction by mass spectrometry. In some aspects, the protein isolated from the active protein fraction may be nidogen-2. In some embodiments, the molecular weight-specific fraction is a >100kDa fraction, a 100kDa to 3kDa fraction, a 3kDa to 1 kDa fraction, or a <1 kDa fraction, particularly a >100kDa fraction.

[0012] Other objects and features will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0014] FIG. 1A shows relative ex vivo glucagon secretion measured in non-diabetic mouse islets (n=3) with different molecular weight fractions from an immortalized brown adipocyte cell line (imBAT) conditioned buffer (larger than 100kDa, between 10OkDa and 3kDa, between 3kDa and 1 kDa, smaller than 1 kDa; 20 pg / mL) at low (yellow, 1 mM) glucose condition.

[0015] FIG. 1 B shows relative glucagon secretion measured in non-diabetic mouse islets (n=4) with >100kDa fraction from embryonic (eBAT, E14-16, n=6), adult (aBAT, n=4) adipose tissue, and imBAT (20 pg / mL) at low (yellow, 1 mM) glucose condition.

[0016] FIG. 1 C shows relative glucagon secretion measured in low (yellow, 1 mM) and high (teal, 11 mM) glucose conditions in both non-diabetic and diabetic mouse (non-diabetic n=30, T1 D Model (NOD) n=5, T2D (Db / Db) model n=3).

[0017] FIG. 1 D shows relative glucagon secretion measured in low (yellow, 1 mM) and high (teal, 11 mM) glucose conditions in both non-diabetic and diabetic human (non-diabetic n=16, T1 D n=1 , T2D n=8).

[0018] FIG. 1 E shows relative glucagon secretion inhibition activity measured at low (1 mM) glucose in non-diabetic mouse islets (n=5) (yellow) plotted with extracellular vesicles (EVs) particles number (black) of size exclusion column (CL- 2B) fractions with CB-100.

[0019] FIG. 1 F shows relative glucagon secretion inhibition activity measured at low (1 mM) glucose in non-diabetic mouse islets (n=5) (yellow) plotted with 205nm (green) and 260nm (red) absorbance of size exclusion column (CL-2B) fractions with CB-100.

[0020] FIG. 1 G shows relative glucagon secretion measured in non-diabetic mouse islets (n=3) with CB-100 with or without acetone protein precipitation or boiling at low (yellow, 1 mM) glucose conditions. Data are presented as Mean ± SEM. Statistical significance was determined using an unpaired t-test. Groups compared for statistical analysis are indicated by the line. *p < 0.05, **p < 0.01 , ***p < 0.001 , ns (p > 0.05).

[0021] FIG. 2A shows a schematic of the experimental design for CB-100 injection in nonobese diabetic (NOD) mice (14-22weeks old). Once NOD mice reached the early stage of hyperglycemia (blood glucose level between 190mg / dL and 230mg / dL), 1 ,5mg / kg body weight (BW) CB-100 subcutaneous injection was done for 7 days. Afterward, weekly blood glucose monitoring was performed using an intraperitoneal glucose tolerance test (IPGTT), an intraperitoneal insulin tolerance test (I P ITT), and metabolic cages studies. After 2 to 8 weeks of monitoring, mice were euthanized, and postmortem tissues (blood, adipose tissue, skeletal muscle, pancreas, and liver) were collected for further analysis.

[0022] FIG. 2B shows weekly non-fasting blood glucose levels measured following 7 days of injection of CB-100 (subcutaneous, 1.5 mg / kg BW, successful; yellow, n = 30 (19 females, 11 males), reverted; blue, n = 3 (2 females, 1 male)) and sham (KRBH, red; n = 18 (male and female)) in NOD mice (14-22 weeks old). Normal non-fasting blood glucose level for NOD mice was indicated with a dotted line (150mg / dL). Grey highlighted days are the days of the CB-100 injections.

[0023] FIG. 2C shows respiratory Exchange Rate (RER) measured in CB-100- treated (yellow, n=4), sham (red, n = 2), and euglycemic control (green, n=2) NOD mice (female). Measurements were taken every 15 minutes over 3 days, with four measurements averaged for each hourly data point.

[0024] FIG. 2D shows IPGTT blood glucose levels measured at 15, 30, 60, 90, and 120 minutes post 2g / kg BW glucose administration to euglycemic control (green, n=8), CB-100-treated (yellow, n=4), and sham-treated (red, n=3) female NOD mice and calculated Area under the curve (AUC). NOD mouse normal non-fasting blood glucose level is marked with a dotted line (~150mg / dL).

[0025] FIG. 2E shows IPITT blood glucose levels measured at 15, 30, 60, 90, and 120 minutes post 0.75 lll / kg BW insulin administration to euglycemic control (green, n=6), CB-100 treated (yellow, n=12), sham-treated (red, n=3) female NOD mice and calculated AUC. NOD mouse normal non-fasting blood glucose level is marked with a dotted line (150mg / dL).

[0026] FIG. 2F shows non-fasting blood plasma glucagon measured in euglycemic control (green, n = 8), sham-treated (red, n=12), successful CB-100 treated (yellow, n=11 ), and failed CB-1OO treated (blue, n=3) female NOD mice.

[0027] FIG. 2G shows non-fasting blood plasma insulin was measured in euglycemic control (green, n = 12), sham-treated (red, n=5), successful CB-100 treated (yellow, n=10), and failed CB-100 treated (blue, n=3) female NOD mice.

[0028] FIG. 2H shows pancreatic glucagon measured in euglycemic control (green, n = 14), sham-treated (red, n=5), successful CB-100 treated (yellow, n=12), and failed CB-100 treated (blue, n=3) female NOD mice, normalized to body weight.

[0029] FIG. 2I shows pancreatic insulin measured in euglycemic control (green, n = 14), sham-treated (red, n=7), successful CB-100 treated (yellow, n=11 ), and failed CB-100 treated (blue, n=3) female NOD mice, normalized to body weight.

[0030] FIG. 2J shows representative immunofluorescence images of pancreas sections for insulin (yellow), glucagon (magenta), and somatostatin (cyan) of euglycemic, sham-treated, and CB-100 treated female NOD mice. Scale bar: 100 pm. Data are presented as Mean ± SEM. Statistical significance was determined using an unpaired t-test. Groups compared for statistical analysis are indicated by the line. For IPITT and IPGTT, blood glucose level of CB-100-treated was compared with sham-treated NOD mice. *p < 0.05, **p < 0.01 , ***p < 0.001 , ns (p > 0.05).

[0031] FIG. 3A shows percentage change of body weight between pre-and posttreatment of euglycemic control (green, n=8), sham-treated (red, n=6), and CB-100 treated NOD mice (yellow, n=16).

[0032] FIG. 3B shows lean and fat mass percentage relative to body weight of euglycemic (green, n = 3), sham (red, n = 3), and CB-100-treated (yellow, n = 4) female NOD mice.

[0033] FIG. 3C shows percentage fat mass relative to the body weight of inguinal white adipose tissue (ingWAT) and brown adipose tissue (BAT) from euglycemic (green, n = 7), sham (red, n = 7), and CB-100-treated (yellow, n = 7) female NOD mice.

[0034] FIG. 3D shows cluster analysis of heatmap in sham (n = 3) and CB-100- treated NOD mice (n = 4) for inguinal white adipose tissue (ingWAT).

[0035] FIG. 3E shows top 5 Reactome and KEGG pathway classification from gene-set-based analysis of significantly upregulated DEGs (Log2FC > 2, p_val <0.01 ) in ingWAT.

[0036] FIG. 3F shows the volcano plot of DEGs in CB-100-treated NOD mice (n=4) compared to sham-treated (n=3) in ingWAT (p_adjust < 0.0005, abs(Log2FC) > 5). Upregulated genes are highlighted in red circles, and downregulated genes are highlighted in blue.

[0037] FIG. 3G shows relative mRNA expression levels of browning marker genes (Ucp1 , Cidea, Zic1 , and Prdm16; relative to euglycemic control) in ingWAT from euglycemic control (yellow, n=5), sham-treated (red, n=3), and CB-100-treated (brown, n=6) NOD mice (female).

[0038] FIG. 3H shows representative histology and immunofluorescence images of ingWAT from euglycemic control, sham-treated, and CB-100-treated NOD mice with H&E and UCP1 (green) with nuclear staining (DAPI, blue). Scale bar: 100 pm.

[0039] FIG. 3I shows relative Ucp1 mRNA expression in control (navy blue) and CB-100 (13.2 pg / mL, 48 hours) treated (purple) 3T3-L1 (white adipocyte) cell line.

[0040] FIG. 3J shows cluster analysis of heatmap in sham (n = 3) and CB-100- treated NOD mice (n = 4) for brown adipose tissue (BAT) with DEseq2; Top 15 differentially expressed genes (DEGs) with significant upregulation (red) and downregulation (blue) (p_adjust < 0.05, abs (Log2FC) > 2, basemean > 50) were represented.

[0041] FIG. 3K shows top 5 Reactome and KEGG pathway classification from gene-set-based analysis of significantly upregulated DEGs (Log2FC > 2, p_val <0.01 ) in BAT of CB-100-treated (n=4) compared to sham-treated (n=3) NOD mice.

[0042] FIG. 3L shows the volcano plot of DEGs in CB-100-treated NOD mice (n=4) compared to sham-treated (n=3) in BAT (p_adjust < 0.05, abs(Log2FC) > 3). Upregulated genes are highlighted in red circles, and downregulated genes are highlighted in blue.

[0043] FIG. 3M shows relative mRNA expression of thermogenic marker genes (Ucp1 , Rbp4, and Cxcl14; relative to euglycemic control) in BAT of euglycemic control (yellow, n=4-10), sham-treated (red, n=4-5) and CB-100 treated (brown, n=3) NOD mice (female). FIG. 3N shows representative histology and immunofluorescence images of BAT from euglycemic control, sham-treated, and CB-100-treated NOD mice with H&E and LICP1 (green) with nuclear staining (DAPI, blue). Scale bar: 100 pm.

[0044] FIG. 30 shows relative mRNA expression of thermogenic marker genes (Ucp1 , Cidea, and Zic1 ) in control (navy blue) and CB-100 (13.2 pg / mL, 48 hours) treated (purple) imBAT cell line. Data are presented as Mean ± SEM. Statistical significance was determined using an unpaired t-test. Groups compared for statistical analysis are indicated by the line. *p < 0.05, **p < 0.01 , ***p < 0.001 , ns (p > 0.05).

[0045] FIG. 4A shows representative immunofluorescence images of the colocalization of GLUT4 (green) with ct-sa rcoglycan (a plasma membrane marker, red) in gastrocnemius muscle tissues of euglycemic control, sham-treated, and CB-100 treated (success and failed) NOD mice. Colocalizations are denoted by arrows Scale bar: 50 pm.

[0046] FIG. 4B shows quantitative analysis of GLUT4 and ct-sa rcoglycan colocalization in euglycemic control (green, n=3), sham-treated (red, n=3), and CB- 100-treated (successful; yellow, n=5 & failed; blue, n=2) NOD mice. Arrows denote colocalizations. Scale bar: 50 pm.

[0047] FIG. 4C shows hepatic glycogen levels measured in euglycemic control (green, n=10), sham-treated (red, n=4), and CB-100-treated (successful; yellow, n=9 & failed; blue, n=2) NOD mice, normalized to tissue weight.

[0048] FIG. 4D shows relative expression levels of insulin-related glucose transporter marker genes (Glucose Transporter Type 4 (Glut4) and Trafficking Regulator of GLUT4 1 (Trargl )) in ingWAT of euglycemic (green n=9), sham- treated (red, n=5) and CB-100-treated (yellow, n=9) NOD mice (female).

[0049] FIG. 4E shows immunofluorescence of GLUT4 (green) and nuclei (blue) in inguinal white adipose tissue (ingWAT) of euglycemic control, sham-treated, and CB-100 treated NOD mice. Scale bar: 20 pm.

[0050] FIG. 4F shows relative glucose consumption in myotubes (C2C12), hepatocytes (AML12), white adipocytes (3T3-L1 ), and brown adipocytes (imBAT) treated without (control, navy blue) or with CB-100 (purple, 52.8 pg / mL) and in the absence (control) or presence of S961 (200 nM) for 24 hours. FIG. 4G shows immunoblot analysis of p-IRS-1Tyr606 / total-IRS-1 and p- AKTSer473 / total-AKT from L6.GLUT4Myc myotube untreated (navy blue, control) or treated with CB-100 (purple, 10pg / mL, 16-18 hours) then spiked in insulin (10nM, 5minutes). Data are presented as mean ± SEM. Statistical significance was assessed using an unpaired t-test, with significance denoted as follows: *p < 0.05, **p < 0.01 , ***p < 0.001 , ns (p > 0.05). The groups compared for statistical analysis are connected by lines.

[0051] FIG. 5A shows a schematic of the experimental design and process for identifying active proteins from CB-100.

[0052] FIG. 5B shows rank order of protein signal intensity correlation in size exclusion chromatography fractions with the glucagon secretion inhibition activity. Reported secreted proteins are indicated in red circles.

[0053] FIG. 5C shows plot of glucagon secretion level with size exclusion chromatography fractions at low glucose (1mM) condition (grey bar) and nidogen-2 protein intensity (red). (For glucagon secretion, the control condition (1 mM and 11 mM) is indicated with the left two dark grey bars. The expected lowest glucagon secretion is determined with the glucagon secretion level at high glucose condition (11 mM) and indicated with the dotted line.

[0054] FIG. 5D shows immunofluorescence image of nidogen-2 (red), LICP1 (green), and nucleus (blue) in fully differentiated brown adipocytes (imBAT) Scale bar: 50 pm (left) and 20 pm (right).

[0055] FIG. 5E shows western blot analysis of nidogen-2 in recombinant nidogen-2 (Leu31-Lys1403) and >100kDa conditioned buffer from embryonic brown adipose tissue (eBAT) and immortalized brown adipocytes (imBAT).

[0056] FIG. 5F shows interpolated molecular weight of nidogen-2 peaks in SEC elution shown in figure 5C using linear regression from log (molecular weight) versus retention factor (Kav) of molecular weight standard mix which includes thyroglobulin (670kDa), y-globulins (150kDa), albumin (44.3kDa), and ribonuclease-A (13.7kDa).

[0057] FIG. 5G shows non-fasting blood glucose levels measured following 7 days of injection of recombinant mouse nidogen-2 (Leu31-Lys1403) (black, subcutaneous, 20mg / kg BW, n = 5) and sham (red, KRBH, n = 3) in NOD mice (female, 18-22 weeks old). Normal non-fasting blood glucose level for NOD mice was indicated with a dotted line (150mg / dL). Grey highlighted days are the days of the nidogen-2 injections.

[0058] FIG. 5H shows relative glucagon secretion measured in low (yellow, 1 mM) and high (teal, 11 mM) glucose conditions in non-diabetic mouse (n=15) pancreatic islets without or with CB-100 (2.5pg / mL) or recombinant mouse nidogen-2 (Leu31- Lys1403) (40ng / mL). Data are presented as Mean ± SEM. Statistical significance was determined using an unpaired t-test. Groups compared for statistical analysis are indicated by the line. *p < 0.05, **p < 0.01 , ***p < 0.001 , ns (p > 0.05).

[0059] FIG. 5I shows relative glucagon secretion measured in low (yellow, 1 mM) and high (teal, 11 mM) glucose conditions in non-diabetic human (n=4) pancreatic islets without or with CB-100 (2.5pg / mL) or recombinant mouse nidogen-2 (Leu31- Lys1403) (40ng / mL). Data are presented as Mean ± SEM. Statistical significance was determined using an unpaired t-test. Groups compared for statistical analysis are indicated by the line. *p < 0.05, **p < 0.01 , ***p < 0.001 , ns (p > 0.05).

[0060] FIG. 5J shows relative glucose consumption measured in C2C12, AML12, 3T3-L1 , and imBAT cell line without (control, navy blue) or with nidogen-2 (orange, 1 pg / mL) (16 hours).

[0061] FIG. 6A shows relative glucagon secretion measured in low (yellow, 1 mM) and high (teal, 11 mM) glucose conditions in non-diabetic mouse (n=7) and human (n=4) dispersed pancreatic islets without or with CB-100 (2pg / mL) and nidogen-2 (2pg / mL for human, 2ng / mL for mouse).

[0062] FIG. 6B shows relative glucagon secretion measured in low (yellow, 1 mM) and high (teal, 11 mM) glucose conditions with or without S961 (1 pM) and CB-100 (2 pg / mL) or Nidogen-2 (40 ng / mL) in mouse (non-diabetic, n=8) pancreatic islets.

[0063] FIG. 6C shows percentage change calculated from cAMP FRET intensity ratio in non-diabetic mouse pancreatic islets (n=5) in low (yellow, 1 mM) and high (teal, 11 mM) glucose conditions before and after treatment (CB-100 (5 pg / mL) or nidogen-2 (40 ng / mL)) with or without S961 (1 pM).

[0064] FIG. 6D shows Ca2+oscillation frequency measured in low (yellow, 1 mM) and high (teal, 11 mM) glucose conditions in non-diabetic mice (n=7) with or without CB-100 (5 pg / mL) or Nidogen-2 (40 ng / mL) and S961 (1 pM). FIG. 6E shows representation of calcium indicator (GCaMP6) fluorescence intensity tracing in individual mouse pancreatic ct-cells in low (yellow, 1mM) and high (teal, 11 mM) glucose conditions with CB-100 (5 pg / mL) or Nidogen-2 (40 ng / mL) and with or without S961 (1 pM). Data are presented as Mean ± SEM. Statistical significance was determined using an unpaired t-test. Groups compared for statistical analysis are indicated by the line. *p < 0.05, **p < 0.01 , ***p < 0.001 , ns (p > 0.05).

[0065] FIG. 7A shows relative insulin secretion measured in low (yellow, 1mM) and high (teal, 11 mM) glucose conditions in both human (non-diabetic n=16, T1 D n=1 , T2D n=7) and mouse (non-diabetic n=30, T1 D model n=5, T2D model n=3) pancreatic islets.

[0066] FIG. 7B shows relative mRNA expression of Cidea and Ucp1 of imBAT without (pink) or with (green) norepinephrine stimulation (10pM, 16 hours).

[0067] FIG. 7C shows relative glucagon secretion in low (yellow, 1mM) and high (teal, 11 mM) glucose conditions in pancreatic islets from non-diabetic mice (n=4) with CB-100 (26.5pg / mL) collected from imBAT without (NE-) or with (NE+) stimulation (10pM, 16 hours).

[0068] FIG. 7D shows relative glucagon secretion measured without (navy blue) or with CB-100 (purple, 5pg / mL) in low (1mM) glucose condition with GLP-1 receptor (GLP-1 R) antagonist (Exendin-3, 50nM) or glucagon receptor (GCGR) antagonist (Adomeglivant, 1 ,5pM) or Somatostatin receptor (SSTR) antagonist (CYN154806, 25nM) or Ephrin A4 receptor (EphA4R) antagonist (KYL, 50pM), insulin receptor (INSR) antagonist (S961 , 1 pM) in non-diabetic mouse pancreatic islets (n=3). Data are presented as Mean ± SEM. Statistical significance was determined using an unpaired t-test. Groups compared for statistical analysis are indicated by the line. *p < 0.05, **p < 0.01 , ***p < 0.001 , ns (p > 0.05).

[0069] FIG. 8A shows CB-100 treatment success (green) and failed (red) percentage of early (female: n=21 , male: n=12, blood glucose (BG) between 190mg / dL and 230mg / dL) and late-stage (female: n=14, male: n=5, >230mg / dL) of hyperglycemia in female NOD mice.

[0070] FIG. 8B shows CB-100 treatment success (green) and failed (red) percentage of early (female: n=21 , male: n=12, blood glucose (BG) between 190mg / dL and 230mg / dL) and late-stage (female: n=14, male: n=5, >230mg / dL) of hyperglycemia in Male NOD mice.

[0071] FIG. 8C shows metabolic cages measured and averaged activity from euglycemic control (yellow, n = 2), sham (red, n = 12), and CB-100-treated (brown, n = 4) NOD mice.

[0072] FIG. 8D shows metabolic cages measured and averaged heat (kcal / h / kg) from euglycemic control (yellow, n = 2), sham (red, n = 12), and CB-100-treated (brown, n = 4) NOD mice.

[0073] FIG. 8E shows metabolic cages measured and averaged VO2 consumption from euglycemic control (yellow, n = 2), sham (red, n = 12), and CB-100-treated (brown, n = 4) NOD mice.

[0074] FIG. 8F shows H&E Staining of liver sections from euglycemic control, sham-treated, and CB-100 treated NOD mice Scale bar: 100 pm.

[0075] FIG. 8G shows weekly non-fasting blood glucose levels following seven days of injection of CB-100 (subcutaneous, 1.5 mg / kg BW; Success, green, n = 3 & failed, red, n=11 ) and sham (KRBH, black, n = 6) female NOD mice from late hyperglycemia (18-22 weeks old, BG >230mg / dL).

[0076] FIG. 8H shows weekly non-fasting blood glucose levels following seven days of injection of CB-100 (subcutaneous, 1.5 mg / kg BW; green, success n=1 & failed, red, n=4) and sham (KRBH, black, n = 12) male NOD mice from late hyperglycemia (18-22 weeks old, BG >230mg / dL).

[0077] FIG. 9A shows relative glucagon secretion was measured in low (yellow, 1 mM) and high (teal, 11 mM) glucose conditions in non-diabetic mice (n=6) without or with nidogen-2 (Nid2). Concentration with a significant inhibition activity was highlighted with red.

[0078] FIG. 9B shows relative glucagon secretion was measured in low (yellow, 1 mM) and high (teal, 11 mM) glucose conditions in non-diabetic mice (n=6) without or with superoxide dismutase 3 (Sod3). Concentration with a significant inhibition activity was highlighted with red.

[0079] FIG. 9C shows relative glucagon secretion was measured in low (yellow, 1 mM) and high (teal, 11 mM) glucose conditions in non-diabetic mice (n=6) without or with complement factor H (CFH). Concentration with a significant inhibition activity was highlighted with red.

[0080] FIG. 10A shows Nidogen-2 fragmentation using MMP3 enzyme cleavage resulting in active fractions 12-15, 22, and 32-33.

[0081] FIG. 10B shows Nidogen-2 fragmentation using endoproteinase Lys C enzyme cleavage resulting in active fractions 13-15.

[0082] DETAILED DESCRIPTION

[0083] The present disclosure is based, at least in part, on the discovery of a protein that improves insulin signaling / function and type 1 diabetes (T1 D). As shown herein, the identification of a brown adipose tissue (BAT)-secreted factor (e.g., Nidogen-2) negatively regulates glucagon secretion. Glucagon is important because it counteracts insulin’s effects on blood sugar levels. Notably, this protein improves glycemic control by improving glucose tolerance and insulin sensitivity.

[0084] Generally, a BAT-conditioned buffer is any medium or solution that has been exposed to BAT. For example, a BAT-conditioned buffer can be generated by incubating BAT cells with Krebs-Ringer bicarbonate HEPES and 11 mmol / L glucose at 37°C for 4 hours. The BAT-conditioned buffer is then removed and filtered through a 0.22^m polyethersulfone filter to remove any bacterial contamination.

[0085] THERAPEUTIC METHODS

[0086] Also provided is a process of treating diabetes in a subject in need thereof by administration of a therapeutically effective amount of brown adipose tissue (BAT)-secreted factor (e.g., BAT-secreted peptide or fragment thereof), which may inhibit or reduce glucagon secretion.

[0087] Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing a metabolic disorder. A determination of the need for treatment will typically be assessed by a history and physical exam consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, chickens, and humans. For example, the subject can be a human subject.

[0088] Generally, a safe and effective amount of a BAT-secreted factor is, for example, that amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects. In various embodiments, an effective amount of a metabolic disorder described herein can substantially inhibit a metabolic disorder, slow the progress of a metabolic disorder, or limit the development of a metabolic disorder.

[0089] When used in the treatments described herein, a therapeutically effective amount of a BAT-secreted factor can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit / risk ratio applicable to any medical treatment, in a sufficient amount to inhibit or reduce glucagon secretion, increase insulin sensitivity, or treat diabetes.

[0090] The amount of a composition described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.

[0091] Again, each of the states, diseases, disorders, and conditions, described herein, as well as others, can benefit from compositions and methods described herein. Generally, treating a state, disease, disorder, or condition includes preventing or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms. A benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or to a physician.

[0092] Administration of a BAT-secreted factor can occur as a single event or over a time course of treatment.

[0093] Treatment in accordance with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for a metabolic disorder.

[0094] A BAT-secreted factor can be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory, or another agent.

[0095] ADMINISTRATION

[0096] Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.

[0097] As discussed above, administration of agents and compositions described herein, such as brown adipose tissue (BAT)-secreted factor and compositions comprising brown adipose tissue (BAT)-secreted factor, can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.

[0098] More specifically, administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 |uim), nanospheres (e.g., less than 1 ^m), microspheres (e.g., 1-100 ^m), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.

[0099] Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.

[0100] Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331 ). Carrier-based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule / agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency, improve the taste of the product; or improve the shelf life of the product.

[0101] Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41 (1 ), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

[0102] Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0103] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0104] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0105] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0106] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0107] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0108] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0109] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

[0110] EXAMPLES

[0111] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0112] EXAMPLE 1: INSULIN-INDEPENDENT REGULATION OF TYPE 1 DIABETES VIA BROWN ADIPOCYTE-SECRETED PROTEINS AND THE NOVEL GLUCAGON REGULATOR NIDOGEN-2

[0113] The current disclosure investigated the mechanisms underlying the restoration of euglycemia following BAT transplant into T1 D mouse models. These findings suggest that the hormone action of proteins in the large (>100kDa) BAT- secreted fraction (termed CB-100) mediates the positive effects of these transplants. Similar to the effects observed with embryonic BAT transplantation, subcutaneous injection of CB-100 maintains sustained euglycemia (FIG. 2B), with a strong correlation between plasma glucagon levels and the success of treatment. Compared to sham and failed CB-100 treatments, successfully treated NOD mice exhibit near-normal plasma glucagon levels and pancreatic glucagon content (FIGs. 2F and 2H), suggesting that CB-100 suppresses hyperglucagonemia and ct-cell hyperplasia. Early-stage T1 D treatment with CB-100, when detectable 0- cells are present, significantly improved the success rate (FIGs. 8A and 8B), suggesting that the preservation of functional -cells might play a role in the longterm recovery of euglycemia. Immunofluorescence analysis of NOD mice supports this mechanism since successful CB-100 treated animals' pancreata exhibit insulin-positive -cells, while those from sham and failed treatment animals do not (FIG. 2J). However, even though the partial restoration of plasma insulin in successful CB-100 treated mice was observed, those levels remained significantly lower than in euglycemic control animals, and there is no significant difference between successful and failed CB-100 treated NOD mice (FIGs. 2G and 2I). Thus, plasma insulin levels and pancreatic insulin content do not correlate with the efficacy of CB-100 treatment, while plasma glucagon levels do, consistent with previous studies showing the therapeutic benefit of suppressing hyperglucagonemia in type 1 diabetes (Gunawardana et al., Am J Physiol Endocrinol Metab, 2015, 308, pp E1043-1055.).

[0114] Within CB-100, nidogen2 was identified as a regulator in reversing hyperglycemia in T1 D (FIG. 5F), suppressing glucagon secretion in pancreatic islets (FIGs. 5G and 5H) and enhancing glucose uptake in adipocytes (FIG. 5J). While full-length nidogen2 suppresses glucagon secretion (FIGs. 5G and 5H), its suppressive effect is not as strong as that observed with CB-100. Both CB-100 and nidogen2 suppress glucagon secretion in dispersed islet cells, indicating a direct modulation of ct-cell function (FIG. 6A). Live cell imaging shows that CB-100 and nidogen2 substantially decrease the intracellular activity of cAMP and Ca2+, and that these effects are attenuated by antagonizing the insulin receptor (FIGs. 6B-6E). As expected by insulin receptor activation, CB-100 and nidogen2 increase glucose uptake in brown adipocytes in vitro (FIGs. 4F and 5J), while CB-100 also shows increased glucose uptake in myotubes and hepatocytes (FIG. 4F). CB-100 improves in vivo glucose uptake in adipose tissue, skeletal muscle, and liver (FIGs. 4A-4E). Upregulation of GLUT4 trafficking in ingWAT (FIGs. 4D and 4E) is also consistent with CB-100-mediated glucose uptake mediated through insulin receptor (Stockli et al., J Cell Sci, 2011 , 124, pp 4147-4159). In all these cases, the effects of CB-100 and nidogen2 are blocked by the antagonism of the insulin receptor. Taken together, these results suggest that nidogen2 plays a major role in the action of CB-100, and the activity of nidogen2 is mediated, at least in part, by the insulin receptor. Nidogen2 has been reported as a basement membrane protein involved in embryo development, basement membrane assembly, tissue regeneration, and interaction with other extracellular matrix proteins (Miosge et al., Histochem J, 2001 , 33, pp 523-530; Patlaka et al., Biochem Biophys Res Commun, 2014, 454, pp 446-452; Zhang et al., Matrix Biol J Int Soc Matrix Biol, 2022, 112, pp 132-154; Zhou et al., Genes Dis, 2022, 9, pp 598-609; and Wolfstetter et al., Dev Camb Engl, 2019, 146, dev168948). In many respects, recombinant full-length nidogen2 fits with the previous embryonic BAT transplant data (Gunawardana et al., Am J Physiol Endocrinol Metab, 2015, 308, pp E1043-1055 and Gunawardana et al., Diabetes, 2012, 61 , pp 674-682) and replicates the actions of CB-100 (FIGs. 5G-5I and 6), but by other measures, its activity is less pronounced or not observed (FIGs. 5H-5J). Cleavage products in CB-100 and eBAT secretions were detected that are smaller than the reported size of nidogen2 (200kDa) (FIG. 5E) (Walker et al., Kidney Int, 1998, 53, pp 1673-1680). This suggests that nidogen2 cleavage products (FIG. 5E) might be the active component or that multiple components of CB-100 are active together.

[0115] BAT loses thermogenic activity and insulin sensitivity during obesity and type 2 diabetes (Maliszewska et al., Int J Mol Sci, 2021 , 22, pp 1530), consistent with the currently presented data showing BAT "whitening", indicated by decreased thermogenic marker gene expression in T1 D development of NOD mice (FIG. 3L). Analysis of differentially expressed genes (DEGs) in BAT revealed the enrichment in thermogenesis (FIG. 3I) and upregulation of Scd1 , Dio2, and Gm445002, which were reported genes to promote adipogenesis, adipocyte browning, and thermogenesis (FIGs. 3I and 3K) (Wang et al., iScience, 2022, 25, 104809; Liu et al., PNAS, 2020, 117, pp 2462-2472; Zou et al., J Lipid Res, 2020, 61 , pp 1589-1604; and de Jesus et al., J Clin Invest, 2001 , 108, pp 1379-1385). However, further quantitative PCR (qPCR) mRNA expression did not demonstrate a notable difference in thermogenesis marker genes (Ucp1 , Rbp4, and Cxcl14) expression compared to euglycemic controls (FIG. 3L). These findings collectively suggest that CB-100's role is more aligned with preventing the whitening of BAT.

[0116] In addition to preventing the whitening of BAT, the current disclosure showed that CB-100 promotes differentiation and browning of white adipose tissue, thereby facilitating better glucose utilization and energy expenditure. Metabolic studies of live animals showed higher oxygen consumption (VO2, VCO2 / VO2) in CB-100 treated compared to sham-treated animals (FIG. 2C, FIG. 8E), while there is no statistical difference in the animals' physical activity or heat expenditure (FIGs. 8C and 8D). These findings suggest that CB-100 enhances metabolic efficiency and increases glucose utilization. In addition to improving metabolic efficiency, recovery of fat mass in CB-100 treated compared to sham- treated mice was observed, with a return the fat mass and body weight seen in euglycemic control NOD mice (FIGs. 3A and 3B). Recovery of healthy white adipose tissue (WAT) in CB-100 treated NOD mice could enhance glycemic control by secreting beneficial adipokines (Khan et al., Scientifica, 2014, 328592) and help prevent fat accumulation in the liver. H&E staining of ingWAT in sham- treated NOD mice showed fibrotic tissue that constrains adipocyte expansion (FIG. 3N). This is expected to promote ectopic fat deposition and result in fat accumulation in other organs like the liver (Kawai et al., Am J Physiol Cell Physiol, 2021 , 320, pp C375-C391 ). Supporting this concept, liver samples from sham- treated NOD mice displayed small-droplet macro-vesicular steatosis (sd-MaS) fat accumulation, while CB-100 treated and euglycemic NOD livers did not (FIG. 8F). Several proline-related genes (Sprrla, Sprr2f, Sprr2a3, and Prr32), Lep, and Scd1 were significantly upregulated in ingWAT of successful CB-100 treated animals (FIGs. 3D and 3F). Proline serves as a primary precursor to extracellular collagen, a major component of the extracellular matrix (ECM) (Patriarca et al., Front Cell Dev Biol, 2021 , 9). Basement membrane (BM)-associated collagens support adipose tissue differentiation and preadipocyte maturation, and ECM molecules are involved in adipose function regulation, such as energy metabolism (Jaaskelainen et al., Biomedicines, 2023, 11 , pp 1412). Thus, increased proline levels could underscore the role of CB-100 in enhancing adipocyte functionality (Patriarca et al., Front Cell Dev Biol, 2021 , 9). Moreover, the GO enrichment analysis indicated that ECM structure and organization, thermogenesis, and fat cell differentiation are enriched pathways (FIG. 3E). Based on these DEGs analysis, CB-100 treatment-mediates recovery of ingWAT through proline-related protein, which improves ECM stability and structure.

[0117] From DEGs analysis, the upregulation of Lep suggests the regulation of the food intake, increasing energy expenditure, and the browning in CB-100 treated mice's ingWAT (Wang et al., Cells, 2019, 8, pp 372 and Park et al., Metabolism, 2015, 64, pp 24-34). Lastly, Scd1 promotes the adipogenesis and browning of the adipocytes (Liu et al., PNAS, 2020, 117, pp 2462-2472 and Liu et al., Biochem Biophys Res Commun, 2024, 696, 149493). A comparable or higher expression of the thermogenic marker genes (Ucp1 , Cidea, Zic1 , and Prdm16) and protein (UCP1 ) in ingWAT of CB-100 treated compared to non-diabetic NOD mice indicated the further browning by CB-100 (FIGs. 3G and 3N). These results collectively suggest that CB-100 treatment not only promotes the recovery of white adipose tissue mass but also induces browning, thereby enhancing energy expenditure and glucose management.

[0118] Overall, the current disclosure reports a critical role of the large (>100kDa) BAT-secreted proteins in promoting glucose homeostasis by regulating glucagon secretion, activating the insulin receptor, preventing the whitening of BAT, and promoting the browning of ingWAT. Importantly, a key protein was identified, nidogen2, which mimics many important actions of the entire CB-100 fraction. The broad metabolic effects of CB-100 and nidogen2 are detailed through in vitro and in vivo assessments, unveiling a new therapeutic strategy for diabetes management beyond insulin-centric paradigms.

[0119] EXAMPLE 2: CHARACTERIZATION OF LARGE BROWN ADIPOCYTE-SECRETED PROTEINS (CB-100)

[0120] Previous studies have demonstrated a decrease in plasma glucagon levels following the transplantation of eBAT into a T1 D mouse model (non-obese diabetic, NOD) (Gunawardana et al., Diabetes, 2012, 61 , pp 674-682). Further, a conditioned buffer from the brown adipocyte cell line (nbat9) inhibited glucagon secretion from pancreatic islets (Hutchens et al., 2015, http: / / etd.library.vanderbilt.edu / etd-08132015-130825). Various properties of the brown adipocyte-conditioned buffer were analyzed to identify the factor responsible for the glucagon secretion inhibition.

[0121] Significant suppression of glucagon secretion in non-diabetic murine islets was seen in low glucose conditions with conditioned buffer from immortalized brown adipocyte (imBAT) with size fractions exceeding 100 kDa, as well as those within the 1 kDa to 3 kDa range (FIG. 1A). The >100kDa fraction exhibited the strongest suppressive effect and was selected as the size fraction (CB-100) for further investigation. CB-100 was collected from incubating adult BAT (aBAT), eBAT, and cultured imBAT cells, and tested for the glucagon inhibition activity. Glucagon secretion-suppressing activity was observed with eBAT and imBAT but not aBAT (FIG. 1B). These results suggest that the glucagon secretion inhibiting CB-100 is more highly secreted in the embryonic stage of BAT compared to mature BAT, which is consistent with previously reported suppression of hyperglucagonemia in T1 D with eBAT transplantation but not aBAT (Gunawardana et al., Am J Physiol Endocrinol Metab, 2015, 308, pp E1043-1055).

[0122] CB-100 suppresses glucagon secretion ex vivo from both mouse and human islets in low glucose settings, regardless of their diabetic status (FIGs. 1E and 1F). However, CB-100 does not affect insulin secretion in either mouse or human pancreatic islets (FIG. 7A).

[0123] Since a hallmark of brown adipocytes is thermogenesis, the glucagon secretion-inhibiting properties of CB-100 were evaluated to see if there is a correlation with thermogenic activity. Norepinephrine (NE) stimulation increases mRNA expression of the thermogenic activity marker gene, Uncoupling Protein 1 (Ucp1 ) and Cell Death Inducing DFFA Like Effector A (Cidea) in imBAT (FIG. 7B), the glucagon secretion inhibiting activity of CB-100 is not changed in NE stimulated and unstimulated imBAT (FIG. 7C).

[0124] Adipose tissue-secreted extracellular vesicles (EVs) have been recognized as important players in intercellular and organ crosstalk (Camino et al., Int J Mol Sci, 2022, 23, pp 10826). It was investigated whether the glucagon secretioninhibiting activity of CB-100 originated from imBAT-secreted EVs. The number of exosome particles did not strongly correlate with the glucagon secretion inhibition activity (FIG. 1D). Instead, peptide bond absorbance at 205 nm shows a better correlation (FIG. 1D), suggesting that the CB-100-mediated suppression of glucagon secretion is mediated by the large proteins (>100 kDa) rather than through EVs. To confirm the protein-like property of the glucagon secretioninhibiting factor, proteins were isolated from CB-100 by acetone precipitation and heated by boiling. The glucagon secretion-suppressing activity was preserved through acetone protein precipitation but abolished by boiling (FIG. 1C), indicating that the glucagon secretion inhibiting factors within CB-100 have a property of a protein. EXAMPLE 3: CB-100 ENHANCES GLUCOSE MANAGEMENT IN A TYPE 1 DIABETES MODEL

[0125] The in vivo impact of CB-100 was investigated using non-obese diabetic (NOD) mice as a T1 D model. Upon reaching the early diabetic stage (between 190 mg / dL and 230 mg / dL), NOD mice received daily subcutaneous injections of CB- 100 (1 .5 mg / kg BW) or sham (KRBH buffer) for seven consecutive days. After 2~8 weeks of monitoring, blood glucose levels, metabolic activity, and post-mortem organ analyses were performed (FIG. 2A).

[0126] Regardless of sex and age, most of the CB-100-treated mice exhibit a significant and sustained reduction in blood glucose levels for up to nine weeks (19 out of 21 mice for a success rate of 90.5%), while sham-treated mice develop severe hyperglycemia (>600mg / dL) within two weeks (FIG. 2B). Notably, administering the treatment during the early stage of T1 D resulted in a higher success rate compared to the later stage (blood glucose levels >230mg / dL, with a success rate of 21.4%) (FIGs. 8A-8B and 8G-8H).

[0127] Mice receiving CB-100 treatment also improve oxygen consumption (VCO2 / VO2) compared to the sham-treated group (FIG. 2C). To evaluate the effects of CB-100 on glucose homeostasis, treated and untreated NOD mice underwent intraperitoneal glucose tolerance test (IPGTT, 2g / kg BW glucose following 6 hours fast) or intraperitoneal insulin tolerance test (IPITT, 0.75 lU / kg BW insulin following 4 hours fast). In the IPGTT, CB-100-treated NOD mice show a lower area under the curve (AUC) than sham-treated NOD mice but remain higher than the non-diabetic control mice (FIG. 2D). In the IPITT, CB-100-treated mice demonstrate a lower AUC than sham-treated diabetic mice and are not significantly different from healthy control animals (FIG. 2E). These findings suggest that CB-100 treatment improves glucose metabolism in type 1 diabetic mice by improving glucose tolerance and insulin sensitivity.

[0128] EXAMPLE 4: CB-100-MEDIATED RESTORATION OF EUGLYCEMIA IS DEPENDENT ON THE SUPPRESSION OF PLASMA GLUCAGON AND IS INDEPENDENT OF INSULIN

[0129] The roles of regulatory hormones in restoring euglycemia was evaluated by comparing the glucagon and insulin hormone profiles among non-diabetic, CB- 100-treated, and sham-treated NOD mice. Compared to the sham-treated NOD mice, CB-100-treated NOD mice demonstrate a decrease in plasma glucagon levels comparable to the non-diabetic NOD (FIG. 2F). A similar trend is observed in pancreatic glucagon contents where non-diabetic and successfully treated CB- 100 NOD mice show significantly lower levels of pancreatic glucagon compared to sham-treated and unsuccessful CB-100-treated NOD mice (FIG. 2H). Insulin levels do not follow a similar pattern. In both blood plasma and pancreatic contents, insulin values are lower for CB-100 treated mice than for non-diabetic NOD (FIGs. 2G and 2I). While CB-100 treated NOD mice exhibit higher insulin levels than the sham-treated mice, these levels are significantly lower than non-diabetic control NOD mice. Notably, there was no significant difference in insulin levels between unsuccessfully and successfully CB-100 treated mice (FIGs. 2G and 2I), which suggests that these low insulin levels are not the determining factor for restoring euglycemia.

[0130] Histological analysis revealed insulin-positive cells in the pancreases of CB- 100 treated NOD mice, though these cells are not as abundant as in non-diabetic controls (FIG. 2J). Additionally, successfully treated CB-100 mice exhibit an improvement in ct-cell hyperplasia, showing a lower ratio of glucagon-positive cells relative to islet size than failed and sham-treated NOD mice (FIG. 2J). Unlike the plasma and pancreatic hormone levels measurements, pancreata from failed CB- 100 treatments do not show insulin-positive cells.

[0131] EXAMPLE 5: CB-100 ENHANCES THE ADIPOCYTE DIFFERENTIATION AND BROWNING IN WHITE ADIPOSE TISSUE

[0132] Given the critical role of adipose tissue in glucose uptake (Chadt et al., Pflugers Arch, 2020, 472, pp 1273-1298), inguinal white adipose tissue (ingWAT) in NOD mice were analyzed. Since the in vivo browning of ingWAT has been reported (Holmes et al., Sci Rep, 2023, 13, 15485), the effects of CB-100 treatment on ingWAT and its potential contributions to glucose homeostasis were assessed. CB-100 treated mice show a higher body weight compared to sham- treated mice, but they do not exceed the weight of non-diabetic control mice (FIG. 3A). Body composition analysis revealed a higher percentage of fat mass in CB- 100 treated mice compared to the sham treatment, with no significant differences in lean mass (FIG. 3B). Normalizing ingWAT mass to total body weight, CB-100- treated mice displayed increased ingWAT mass (FIG. 3C). Hematoxylin and eosin (H&E) staining of control and CB-100 treated ingWAT showed unilocular lipid droplets, while sham-treated diabetic controls showed adipose tissue fibrosis (FIG. 3N).

[0133] The top 15 upregulated and downregulated differentially expressed genes (DEGs) in CB-100-treated ingWAT versus sham-treated ingWAT revealed a distinct heatmap (FIG. 3D). Analysis of the top 10 upregulated Gene Ontology (GO) enrichment analysis highlights extracellular matrix organization, adipocyte differentiation, and thermogenesis (FIG. 3E). A volcano plot applying specific cutoffs (p-value < 0.0005, |log2 fold change] > 5) identified 61 significantly upregulated and 5 downregulated genes (FIG. 3F). Noteworthy genes, such as Small Proline-rich Proteins 1A, 2F, and 2A3 (Sprrla, Sprr2f, and Sprr2a3), Proline- rich 32 (Prr32), Leptin (Lep), and Stearoyl-coenzyme A desaturase 1 (Scd1 ) are upregulated (FIGs. 3D and 3F). ingWAT qPCR mRNA expression analysis (FIG. 3G) showed a high expression of browning marker genes (Ucp1 , Cidea, Zic Family Member 1 (Zic1 ) and PR / SET Domain 16 (Prdm16)) in CB-100 treated compared to sham-treated NOD mice. Immunofluorescence staining of ingWAT showed increased LICP1 in CB-100-treated NOD compared to euglycemic control and sham-treated NOD (FIG. 3N).

[0134] To test if CB-100 directly promotes the browning of white adipocytes, the effect of CB-100 on browning was tested in vitro. 3T3-L1 cells treated with CB-100 for two days demonstrated increased Ucp1 mRNA expression (FIG. 3H). These results collectively indicate that CB-100 treatment promotes adipocyte differentiation and the browning of white adipose tissue, which likely contributes to enhanced glucose homeostasis.

[0135] EXAMPLE 6: CB-100 PREVENTS THE WHITENING OF BROWN ADIPOSE TISSUE

[0136] BAT is recognized for its role in glucose homeostasis through increased energy expenditure and enhanced insulin sensitivity (Maliszewska et al., Int J Mol Sci, 2021 , 22, 1530). In addition to the reported BAT whitening in the context of obesity and type 2 diabetes (Lapa et al., Sci Rep, 2017, 7, 16795), a reduction in BAT thermogenic activity marker genes (Ucp1 , Retinol binding protein 4 (Rbp4), and Chemokine (C-X-C motif) ligand 14 (Cxcl14)) was observed with the development of T1 D in NOD mice (FIG. 3L).

[0137] Although BAT mass is similar between CB-100 treated and sham-treated NOD mice (FIG. 3C), the DEGs heatmap reveals distinct expression patterns in endogenous BAT of CB-100 treated compared to sham-treated NOD mice (FIG. 3I). Reactome and KEGG pathway analyses of CB-100 treated BAT compared to sham-treated revealed enrichment thermogenic activity pathways (FIG. 3J). A volcano plot with specific cutoffs (p-value < 0.05, |log2 fold change] > 3) showed 16 significantly upregulated and 22 downregulated genes (FIG. 3K). Noteworthy genes, such as iodothyronine deiodinase 2 (Dio2), Scd1 , and LncRNA Gm445002 are upregulated (FIGs. 3I and 3K).

[0138] Although gene expression analysis indicates enhanced thermogenic activity in BAT of CB-100 treated NOD mice compared to sham, there are no significant differences in the expression levels of thermogenesis marker genes (Ucp1 , Rbp4, and Cxcl14) between euglycemic control and CB-100-treated NOD mice (FIG. 3L). Consistent with the in vivo data, in vitro experiments with immortalized brown adipocytes (imBAT) treated with CB-100 show no significant changes in thermogenesis-related gene expressions (Ucp1 , Cidea, and Zic1 ) compared to untreated (FIG. 3M).

[0139] H&E staining of BAT showed enlarged lipid droplets in sham-treated NOD compared to euglycemic control and CB-100 treated NOD mice, while there was no noticeable difference in lipid droplets between euglycemic control and CB-100 treated NOD mice (FIG. 3N). Immunofluorescence staining of BAT from CB-100 treated NOD mice reveal a higher LICP1 expression and the presence of multilocular lipid droplets compared to the sham-treated NOD's BAT (FIG. 3N). No significant differences were observed compared to non-diabetic control mice (FIG. 3N). These findings suggest that CB-100 treatment prevents the reduction of BAT thermogenic activity due to the development of T1 D in NOD mice.

[0140] EXAMPLE 7: CB-100 ENHANCES GLUCOSE UPTAKE IN SKELETAL MUSCLE, LIVER, AND ADIPOSE TISSUE

[0141] Immunofluorescence of gastrocnemius muscles from non-diabetic, CB-100 treated, and sham-treated NOD mice revealed higher colocalization of GLUT4 and ct-sa rcoglycan in CB-100 treated mice (FIGs. 4A and 4B). This finding indicates enhanced GLUT4 translocation to the plasma membrane, facilitating increased glucose uptake. To determine the role of liver glucose homeostasis in CB-100 action, liver glycogen levels were measured, which displayed higher liver glycogen levels in successful CB-100 treated mice than in sham-treated or failed CB-100 treated animals (FIG. 4C).

[0142] Immunofluorescence of adipose tissue (BAT and ingWAT) from nondiabetic, CB-100 treated, and sham-treated NOD mice revealed increased expression of GLUT4 in CB-100 treated NOD's adipose tissue compared to sham- treated (FIG. 4D). While ingWAT's GLUT4 expression of CB-100 treated NOD is higher than euglycemic and sham-treated NOD, BAT does not show as high expression of GLUT4 as euglycemic control (FIG. 4D). Moreover, relative gene expression in ingWAT showed upregulation of insulin-regulated glucose transporter (GLUT4) (Stockli et al., J Cell Sci, 2011 , 124, pp 4147-4159) trafficking- related genes (Trafficking regulator of GLUT4 (SLC2A4) 1 (Trargl ) and Solute Carrier Family Member (Slc2a4) (Duan et al., Biochem J, 2022, 479, pp 1237- 1256)) in CB-100 treated NOD compared to the sham treatment (FIG. 4E). Successful CB-100 treatment correlates with higher expression of these genes than failed CB-100 treatment (FIG. 4E).

[0143] In vitro analyses of C2C12 myotubes, AML12 hepatocyte, 3T3-L, and imBAT cells showed enhanced glucose uptake with CB-100 and diminished effects following exposure to the insulin receptor antagonist S961 (FIG. 4F). These findings collectively suggest that CB-100 treatment enhances glucose uptake in adipose tissue, skeletal muscle, and liver, which leads to improved glucose homeostasis.

[0144] EXAMPLE 8: IDENTIFICATION OF NIDOGEN2 AS A BROWN ADIPOCYTE-SECRETED GLUCAGON SECRETION REGULATOR PROTEIN

[0145] Expanding upon the discovery of CB-100, a series of separation techniques was used to identify the glucagon secretion-inhibiting proteins underlying these effects. The CB-100 fraction was directed to anion-exchanged and size-exclusion chromatography (SEC) and fractions from SEC were evaluated by mass spectrometry and correlated with their ability to inhibit glucagon secretion (FIG. 5 A)

[0146] Mass spectrometry revealed that nidogen2 is strongly correlated with glucagon secretion inhibition (FIG. 5B). Pearson's correlation coefficient versus protein rank showed the top candidate secreted proteins (FIG. 5C), which was tested for glucagon inhibition (FIGs. 9A-9C). Based on its robust and consistent inhibition of glucagon secretion from murine and human islets, nidogen2 was identified for future investigation. Immunofluorescence showed intracellular nidogen2 in fully differentiated imBAT (FIG. 5D), and western blot analysis confirms nidogen2 in the conditioned buffer from both eBAT and imBAT (FIG. 5E), although the cellular secretion showed cleaved nidogen2 fragment rather than only the full-length protein.

[0147] When recombinant mouse nidogen2 (Leu31 -Lys1403) was injected into the early stage of T1 D NOD mice, nidgoen2-treated NOD mice showed the recovery of euglycemia (FIG. 5F). Moreover, recombinant mouse nidogen2 suppressed glucagon secretion to mouse and human pancreatic islets (FIGs. 5G and 5H). These findings identify nidogen2 as a brown adipocytes-secreted regulator protein for hyperemia and glucagon secretion.

[0148] EXAMPLE 9: REGULATION OF INTRACELLULAR MESSENGERS OF GLUCAGON SECRETION BY CB-100 AND NIDOGEN2 IN PANCREATIC A-CELLS

[0149] To investigate the mechanisms underlying glucagon secretion inhibition by CB-100 and nidogen2, ct-cell signaling pathways were examined. Both CB-100 and nidogen2 inhibit glucagon secretion from dispersed islet cells from mouse and human islets under low and high glucose conditions (FIG. 6A). These data indicate that CB-100 and nidogen2 influence a-cells directly and not indirectly through other islet cell types.

[0150] To determine if specific receptors on pancreatic a-cells mediate the inhibition, antagonists were tested for the glucagon receptor (GCGR), GLP-1 receptor (GLP1 R), somatostatin receptor (SSTR), EphA4 receptor (EphA4R), and insulin receptor (INSR) on CB-100 or nidogen2 activity. No changes were observed in these activities after antagonizing GCGR, GLP-1 R, SSTR, or EphA4R (FIG. 7D). However, the INSR antagonist (S961 ) diminished the inhibitory effects of CB-100 and nidogen2 under low and high glucose conditions (FIG. 6B).

[0151] Intracellular cAMP and Ca2+activity were measured by live cell imaging of a-cells in murine islets. CB-100 and nidogen2 inhibited both intracellular cAMP and Ca2+activity compared to control (FIGs. 6C-6E). The inhibition of intracellular cAMP and Ca2+activities is blocked by the insulin receptor antagonist, S961 (FIGs. 6C-6E). These results indicate that the suppression of glucagon secretion by CB-100 and nidogen2 in pancreatic a-cells is mediated at least in part through an insulin receptor-dependent manner. EXAMPLE 10: SEARCH NIDOGEN-2 FRAGMENTATION FOR ACTIVE PEPTIDES USING MMP3 ENZYME CLEAVAGE SHOWS ACTIVE FRACTIONS 12-15, 22, AND 32- 33

[0152] The search for Nidogen-2 fragmentation for active peptides using MMP3 enzyme cleavage or endoproteinase Lys C enzyme cleavage shows active fractions 13-15 (FIGs. 10A and 10B).

[0153] EXAMPLE 11: METHODS

[0154] Cell Culture

[0155] Immortalized brown preadipocyte (imBAT, derived from stromal vascular fraction of brown adipose tissue) cells were provided by M. Christian, Ph.D. (University of Warwick, Division of Metabolic and Vascular Health (Rosell et al., Am J Physiol Endocrinol Metab, 2014, 306, pp E945-964)). Preadipocyte differentiation was induced by culturing with differentiation media (DMEM / F12 + GlutaMAX, 10% FBS, 1 % Penicillin-streptomycin, 170nM insulin, 250nM Dexamethasone, 0.5mM IBMX, 0.1 nM T3, 125uM Indomethacin) at 33°C for 48 hours. Differentiated imBAT cells were then cultured with maintenance media (DMEM / F12 + GlutaMAX, 10% FBS, 1 % Penicillin-streptomycin, 170nM insulin, 0.1 nM T3) at 37°C for 8 days, as previously described (Rosell et al., Am J Physiol Endocrinol Metab, 2014, 306, pp E945-964; Debevec et al., Mol Endocrinol, 2007, 21 , pp 1581-1592; Hallberg et al., Mol Cell Biol, 2008, 28, pp 6785-6795; and Morganstein et al., J Lipid Res, 2008, 49, pp 679-685).

[0156] Myotube C2C12 cells were cultured with DMEM, 20% FBS, 1 % Penicillin- streptomycin to 60-70% confluency. Once it reached the desired confluency, cells were differentiated (DMEM, 2% Horse serum, 1 % Penicillin-streptomycin, change media every 2-3 days) for 10 days before being ready for the experiment as previously described (Kumar et al., eLife, 2020, 9, e58941 ).

[0157] 3T3-L1 adipocytes (ATCC) were differentiated (DMEM, 10% BCS, 1 % Penicillin-streptomycin, 0.5mM IBMX, 1 uM Dexamethasone, 170nM Insulin) and maintained (DMEM, 10% BCS, 1% Penicillin-streptomycin, 170nM Insulin) as described (Morrison et al., Adipocyte, 2015, 4, pp 295-302).

[0158] Experimental Animals

[0159] All mouse works were performed under the approval of the Washington University Institutional Animal Care and Use Committee. 8-14-week-old male and female wild-type C57BL / 6 (Jackson Laboratory) were used for the islet isolation. For pancreatic ct-cells calcium imaging, mice with ct-cells expressing GCaMP6f (GCG-iCre-GCaMP6 mice) have been previously described (Ng et al., Diabetes, 2022, 71 , pp 2384-2394). For cAMP measurement in ct-cells, mice expressing red fluorescent protein in pancreatic ct-cells (ctRFP mice) have been previously described (Le Marchand et al., J Biol Chem, 2010, 285, pp 14389-14398). For the CB-100 injection experiment, NOD / ShiLtJ (Jackson Laboratory) was ordered at 9- week-old, and blood glucose level was checked weekly to monitor the status of the development of diabetes.

[0160] Non-diabetic and Type 2 diabetic human islets were received from the Integrated Islet Distribution Program (IIDP), and type 1 diabetic human islets were received from the Laboratory of Al Powers at Vanderbilt University Medical Center. Received islets were cultured in islet media overnight before use.

[0161] Pancreatic islet isolation and culture

[0162] Murine pancreatic islets were isolated by digesting the pancreas from C57BL / 6 mice (10-14 weeks old) with 12mg Collagenase P (Roche) at room temperature with rotation for 35 minutes. After digestion, islets were washed twice with 10mL of G-Solution (HBSS with 1 % BSA) by centrifuging at 300g for 3 minutes and discarding the supernatant. After washing, pellets containing islets were manually picked or isolated through density gradient centrifugation.

[0163] For manual picking, the pellet was reconstituted into islet media (RPMI 1640 with 10% FBS, 1 % Penicillin-streptomycin, 20 mM HEPES, 11 mM glucose) and placed into petri dishes. Under a dissection microscope, islets were picked using a 10uL pipette and placed in a new dish containing 10mL of islet media for overnight recovery at 37°C.

[0164] For density gradient islets isolation, the pellet was reconstituted into 10mL of G-Solution and filtered into a 50m L conical tube through a metal strainer (500pm), and an additional 10mL of G-Solution was added to the filtrate. Islets were pelleted by centrifuge at 300g for 3 minutes, and the supernatant was removed. The pellet was resuspended in 15mL of histopaque-1100 (45.45% Histopaque 1077, 54.54% Histopaque 1119, room temperature) and centrifuged at 290g for 20 minutes. The supernatant was transferred to a new 50m L tube with 25mL of G-solution. Islets were pelleted by centrifuging at 450g for 5 minutes, and the supernatant was removed. Islet pellet was washed with 10mL of G-solution by centrifuging at 300g for 3 minutes. The washed pellet was resuspended to 5m L of islet media and transferred to a petri dish with 5mL of islet media. Islets were incubated at 37°C overnight for recovery before the experiment.

[0165] For dispersing islets, isolated islets were washed with HBSS (without Ca2+or Mg2+) and dissociated with 0.5mL of Accutase (innovative cell technology) for 10 minutes at 37°C with intermittent pipetting every 5 minutes. After dissociation, 0.5mL of islet media is added to stop the Accutase activity.

[0166] Hormone Secretion Assays

[0167] 5-7 pancreatic islets or dispersed islets were seeded into each well of a half-area 96-well plate (Coming Costar) coated with 100ug / mL Rh-Laminin 521 (Gibco) and incubated with islet media overnight before experiments. Islets were briefly washed once with equilibrium media (RPMI 1640, 20mM HEPES, 0.1 % BSA, 2mM glucose), and islets were incubated with 50uL of equilibrium media for 45 minutes at 37°C. After incubation, the equilibration buffer was saved, and islets were incubated with islet secretion buffer (RPMI 1640, 20mM HEPES, 0.1 % BSA) with low (1 mM) or high (11 mM) glucose with the treatment condition for 1 hour at 37°C. After incubation, the secretion buffer was saved. Glucagon and insulin were measured in duplicate with Lumit™ Glucagon and Insulin Immunoassay (Promega), respectively. For normalizing the data with respect to islet mass, the secreted glucagon level was calculated by dividing the glucagon level in the secretion sample by the equilibration value that was divided by the median value of all glucagon levels in equilibration samples.

[0168] For relative glucagon and insulin secretion level calculation, media for low glucose (1 mM, for glucagon secretion) or high (11 mM, for insulin secretion) was calculated, and secreted glucagon or insulin level was divided by this median value to get ‘relative secretion’ value.

[0169] Conditioned Buffer collection and fractionation

[0170] Fully differentiated imBAT cells were briefly washed with PBS and incubated with Krebs-Ringer bicarbonate HEPES (KRBH) buffer (128.8 mM NaCI, 4.8 mM KCI, 1.2 mOM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCI2, 20 mM HEPES, and 5 mM NaHCOs, pH 7.4) with 1 1 mM glucose at 37°C for 4 hours and then collected. The collected conditioned buffer was filtered with a 0.22um PES filter (Genesee Scientific) and fractionated with Ultracel 100kDa ultrafiltration Discs (Millipore) using Amicon Stirred Cells system (Millipore) using nitrogen gas pressure (1 Opsi). The fraction with a molecular weight bigger than 10OkDa (CB- 100) was collected and protein concentration was measured using Pierce Quantitative Peptide Assays (Thermo Scientific) for further analysis and experimentation.

[0171] For further fractionation, the CB-100 served as the starting material for downstream liquid chromatography experiments. Fractions eluted from an anion- exchange column were collected using an AKTA pure 25M system equipped with a multi-wavelength detector. The column (Cytiva, HiScale 26 / 20) was packed inhouse with Capto Q resin (Cytiva, 17531602). During the binding step, the chromatography method employed buffer A (100 mM Tris, pH 8.0), while the elution step utilized a linear gradient from 0% to 50% of buffer B (100 mM Tris, pH 8.0, 1 M NaCI). To evaluate their biological activity, the fractions were subjected to a test wherein glucose was used to stimulate glucagon secretion from pancreatic islets. The active fractions were subsequently concentrated and processed through a pre-packed size-exclusion column (Superdex 200 Increase 10 / 300 GL), with the running buffer consisting of 100 mM Tris, pH 8.0. Once again, all the fractions were evaluated for their biological activity.

[0172] Mass Spectrometry Sample Preparation

[0173] Size exclusion chromatography (SEC) fractions were diluted 1 :1 with 8 M Urea, 75 mM NaCI, 50 mM Tris, pH 8. The SEC fractions were then reduced with 5 mM dithiothreitol for 45 minutes at 37°C, followed by alkylation for 30 minutes at room temperature with 50 mM chloroacetamide. Then, samples were diluted to 1 M Urea with 50 mM Tris, pH 8, and digested with 0.4 ug of trypsin overnight at 37°C at 800 RPM on a thermomixer. After digestion, samples were quenched with trifluoroacetic acid and dried in a speed-vac. Each SEC fraction was then desalted using C18 ZipTips (Millipore Sigma) prior to LCMS analysis.

[0174] Proteomics data acquisition and analysis

[0175] Digested SEC fractions were separated via reverse-phase nano-HPLC using an RSLCnano Ultimate 3000 (Thermo Fisher Scientific). The mobile phase consisted of water + 0.1 % formic acid as buffer A and acetonitrile + 0.1 % formic acid as buffer B. Peptides were loaded onto a pPAC™ Trapping column (PharmaFluidics) and separated on a 50 cm pPAC™ column (PharmaFluidics) operated at room temperature and flowing at 750 or 300 nL / min using a segmented gradient as follows: 5 min from 2-12% buffer B at 750nl / min, then 42 min from 12-20% B at 300nl / min, and finally 16 min from 20-40% B at 300nl / min. Mass spectrometry analysis was performed on an Orbitrap Eclipse (Thermo Fisher Scientific) using a data-independent acquisition method. MS1 scans were obtained at 60,000 resolution in the Orbitrap with a scan range from 390-1010 m / z, 60 ms max injection time, and an automated gain control (AGO) target of 1e6. Cycles of 38 DIA MS2 scans were obtained at 30,000 resolution in the Orbitrap, using 16 m / z wide quadrupole isolation windows, 54ms max injection time, 5e5 AGC target, and 30% higher-energy collision dissociation energy.

[0176] After data acquisition, peptide and protein identification and quantification were performed by DIA-NN (1 .8.2 beta 27). An in silico tryptic spectral library was created for the mouse SwissProt proteome (downloaded 8 / 27 / 19), along with common contaminants for a total of 17,194 protein entries. Carbiomothemylated cysteine was used as a static modification, one oxidized methionine was set as a variable modification, and one missed cleavage was allowed. Heuristic protein inference was enabled, all protein and gene identifications were globally controlled to an FDR of 0.01 , quantification was performed with QuantUMS (high precision), and normalization was disabled. Secreted protein annotations were downloaded from The Human Protein Atlas48. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD051972.

[0177] Exosome isolation and particle counting

[0178] Sepharose CL-2B particles (Sigma-Aldrich, CL2B300) were packed a few hours before use. CL-2B slurry was prepared by mixing 75% beads with 25 % of 50% aqueous isopropanol. A 10mL centrifuge column (Pierce, 89898) was prepared by rinsing the column with 50% isopropanol and slowly adding 15mL of CL-2B slurry to settle down and condense as the filter retains the beads. Once beads were settled, the column was washed with 13mL of 50% isopropanol twice and DI water once. CL-2B size exclusion column was conditioned by running 50mL of PBS through the column. For separation of the exosome, 1 mL of CB-100 was added to the conditioned CL-2B column, and elution was fractionated (0.5mL) by adding 1 .5 column volume of PBS. Each fraction was tested for glucagon secretion inhibition activity, counted for exosome particle number with NTA ZetaView, and measured for absorbance at 205nm with a Nanodrop.

[0179] Live-cell imaging Pancreatic islets were seeded onto an 8-well-chambered cover glass system (Cellvis) coated with Rh-laminin521 (Gibco, 2ug / cm2) and incubated in islet media for 3 days prior to experiments.

[0180] For Ca2+imaging, the seeded islets were placed in a Zeiss LSM 880 confocal microscope with a heated, CO2-controlled microscope stage (37°C, 5% CO2) and a Plan-Apochromat 63X 1 .4 NA oil immersion objective. The islet media was switched to imaging media (KRBH with 0.1 % BSA) with 2 mM glucose for 30 minutes. For secretion, the buffer was changed to islet media with or without insulin receptor antagonist (S961 ) (Phoenix Pharmaceuticals inc) in 1 mM or 11 mM glucose and incubated for 20 minutes at 37°C, 5% CO2. After incubation, an initial baseline time course image was acquired for 15 minutes. After baseline image acquisition, the buffer was switched to imaging media with either 1 mM or 11 mM glucose with treatment (CB-100: 5ug / mL, nidogen2 (40ng / mL, Biotechne R&D Systems)) and incubated for 5 minutes. After 5 minutes, the treatment image time course was acquired for 15 minutes. GCaMP6f fluorescence was acquired with 488nm laser excitation, detected at an emission range of 490-597nm with the spectral detector as described (Ng et al., Diabetes, 2022, 71 , pp 2384-2394).

[0181] For cAMP FRET imaging, EpacSH187-cAMP FRET biosensor (mTurquoise2A_Epac(CD,A DEP,Q270E)_cpVenus_Venus) was used (Klarenbeek et al., PLOS ONE, 2015, 10, e0122513). The sensor was packaged in adenovirus for delivery, and virus particles were concentrated to a titer of 3.28x1012particles / mL. Adenovirus stock is directly added to islet culture media (3.28x1011particles / mL) and cultured for 3 days at 37°C, 5% CO2 prior to the experiment. The seeded islets were placed in a Zeiss LSM 880 confocal microscope with a heated, CO2-controlled microscope stage (37°C, 5% CO2) and a Plan-Apochromat 20X / 0.2 M27 objective. Islets were equilibrated for 30 minutes in imaging media with 2mM Glucose. The baseline image was acquired after incubating islets with KRBH with 0.1 % BSA with or without S961 (Phoenix Pharmaceuticals Inc) in 1 mM or 11 mM glucose for 30 minutes. Then, the buffer was switched to imaging media with either 1 mM or 11 mM glucose with treatment (CB-100: 5ug / mL, nidogen2 (Biotechne R&D Systems)) and incubated for 30 minutes prior to acquiring a treatment image. RFP ct-cells were imaged with 561 nm laser excitation to mark the RFP ct-cells. FRET signals for acceptors and donors (mVenus, mTurquoise) was measured with LSM 880 spectral detector mode with 405 nm excitation and detected at an emission range of 445-650 nm. Using the entire emission spectrum image, the acceptor and donor's signals were acquired through linear unmixing with the ZEISS ZEN imaging software. For analysis, the percentage change of the Donor-to-acceptor ratio was calculated before and after treatment (the FRET signal is inversely proportional to the concentration of cAMP).

[0182] Western Blotting

[0183] Proteins were mixed with 4x protein sample loading buffer (Li-COR) and boiled for 5 minutes at 95°C. Prepared samples were used for electrophoresis with 4-20% protein gel (Bio-Rad) and transferred to a PVDF membrane (Millipore). After blocking for 1 hour with TBS-T (TBS with 0.1 % Tween20) with 2% BSA, the membrane was incubated with primary antibody (Nidogen2 polyclonal Rabbit (1 :1000)) in TBS-T with 1 % BSA at 4°C overnight with shaking. Then, the membrane was washed with TBS-T 3 times and incubated with a secondary antibody (Goat anti-rabbit IRDye 680 (1 :2000)) in TBS-T with 1 % BSA for an hour at room temperature with shaking. The membrane was washed with TBS-T 3 times (10 minutes each) and visualized by Li-COR Odyssey M Imager (Li-COR).

[0184] CB100 and Nidogen2 Injection in vivo experiment

[0185] NOD mice with blood glucose levels between 190 mg / dL and 230 mg / dL received daily injections of CB-100 (1.5 mg / kg BW, nape of the neck, subcutaneous) or sham (KRBH) for 7 consecutive days on the same time. Blood glucose levels were measured weekly with a blood glucose meter (Glucocard Vital). After 2~8 weeks of blood glucose monitoring, mice underwent IPGTT, IP ITT, and metabolic activity studies, and were euthanized for tissue sample collection.

[0186] Glucose and Insulin Tolerance tests

[0187] Mice underwent a 6-hour fast before IPGTT or a 4-hour fast before IP ITT. After a brief isoflurane anesthesia, blood glucose levels and body weight were recorded, and mice received intraperitoneal injections of 0.75 lU / kg BW Humulin R (Lilly) (for I P ITT) or 2g / kg BW glucose (for IPGTT). After the injection, blood glucose levels were measured at 15, 30, 60, 90, and 120 minutes.

[0188] Body composition Analysis

[0189] After measuring the body weight, lean and fat mass was measured with Echo MRI (EchoMRI) without anesthesia as previously described (Thierry et al., PLOS ONE, 2014, 9, e112450). Two measurements were averaged to calculate the % mass with the body weight. Metabolic studies

[0190] Mice were housed in the TSE Phenomaster system (TSE Systems) overnight before the data was collected. After overnight acclimation, metabolic activities (heat, VO2, and activity) data was collected as previously outlined (McAllan et al., PLOS ONE, 2014, 9, e88904). Metabolic activities were measured every 15 minutes for 3 days, with four measurements averaged for each hourly data point.

[0191] Tissue sample collection from in vivo study

[0192] Following weekly blood glucose measurements around 1 PM, animals were euthanized, and blood plasma was collected via cardiopuncture. The collected blood was mixed with 15 pL of EDTA and centrifuged at 2000g for 15 minutes at 4°C. The supernatant (blood plasma) was then collected and stored at -80°C for further analysis. The entire pancreas, inguinal WAT, BAT, and gastrocnemius muscle were collected, while the liver underwent perfusion with PBS before collection.

[0193] Hormone analysis

[0194] Glucagon and insulin were measured with ELISA kit (Crystal Chem) for blood plasma hormone measurement. For Pancreatic hormone analysis, pancreatic hormones were extracted to 2m L of acid ethanol per mouse following previously reported methods (Gunawardana et al., Diabetes, 2012, 61 , pp 674- 682). Pancreatic insulin and glucagon levels were measured with Immunoassay kits (Promega). If the entire pancreas was used to extract pancreatic hormone levels, the mice body weight was used for normalization. If a portion of the pancreas was used, tissue weight or protein concentration was used for normalization.

[0195] Histology

[0196] For histological analysis, the collected tissues were briefly washed with cold PBS, fixed in 10% Neutral buffered formalin for 2 days at room temperature, and transferred to 70% ethanol for at least 2 days. For hematoxylin-eosin (H&E) staining and immunofluorescence (IF) staining, neutral buffered formaldehyde- fixed tissues were submitted for sectioning to the Anatomic and Molecular Pathology (AMP) Core Labs at Washington University in St. Louis for the paraffin embedding and sectioning into 5um cross-sections. H&E staining was also performed by the AMP core. For IF staining, sections were deparaffined (xylene, 10min, 5min), rehydrated (100% EtOH (10min), 90% EtOH (5min), 70% EtOH (5min), PBS (5min)), boiled with antigen retrieval (Diva Decloaker, 15min), permeabilized with PBS-T (1 hour), and blocked (PBS-T with 2% BSA, 1 hour). For adhered cell IF imaging, cells were fixed with 4% PBS buffered formalin for 30 minutes and permeabilized with 0.1% Triton X-100 in PBS for 15 minutes. Then cells were blocked with 2% BSA in PBS with 0.05% Tween-20 for 30 minutes.

[0197] For immunofluorescence staining, the sample was incubated with primary antibodies diluted in a blocking buffer at 4°C overnight. After washing slides with PBS 3 times (10min, 5min, 5min), slides were incubated with secondary antibodies in room temperature for 1 .5 hours, DAPI staining for 5 minutes, washed, and mounted with prolong glass antifade Mountant (Invitrogen). The image was acquired with Carl Zeiss LSM 880 Airyscan two-photon confocal microscope.

[0198] RNA Sequencing and Analysis

[0199] RNA from the tissue or cells was isolated using RNeasy Lipid mini (Qiagen). Isolated RNA samples were submitted to The Genome Access Technology Center at Washington University in St. Louis for the RNA sequencing. All gene counts were imported into the R DESeq2 package. For cluster analysis, ComplexHeatmap package was used (top 15 most upregulated or downregulated genes were represented; cutoff: p_adjust < 0.05, abs (Log2FC) > 2, basemean > 50). For enrichment analysis, clusterProfiler package using biomaRt and pathfindR package (Log2FC > 2 cutoff) were used, and top 10 more enriched processes were represented. For over-representation analysis, DEGs entrezgene from BAT (CB- 100 treated vs sham; Log2FC >2) was entered to ConsensusPathDB-mouse (http: / / cpdb.molgen.mpg.de / MCPDB)53for gene set over-representation analysis (Reactome and keg pathway, minimum overlap with input list :2, p-value cutoff :0.01 ). For gene ontology categories, level 2 and level 3 were selected for all biological, molecular, and cellular components with p-value cutoff of 0.01. Top 5 reactome and KEGG-enriched pathways are represented. To generate a volcano plot, the EnhancedVolcano package was used. For ingWAT, p_adjust < 0.0005, abs(Log2FC) > 5 cut-off was used. For BAT p_adjust < 0.05, abs(Log2FC) > 3 cutoff was used.

[0200] Quantitative PCR analysis

[0201] RNA was extracted from tissues or cells using either the RNeasy lipid mini kit (Qiagen) or TRIzol reagent (Invitrogen), and cDNA was synthesized using the Superscript VILO cDNA synthesis kit (Invitrogen). Quantitative real-time polymerase chain reaction (PCR) was conducted using the Powerllp SYBR Green master mix (Applied Biosystems) and the ViiA7 Real-Time PCR System (Applied Biosystems). The comparative cycle-threshold (Ct) method with 18S ribosomal mRNA expression as the housekeeping gene was used for the result analysis.

[0202] In vitro Glucose consumption assay

[0203] Pre-existing culture media was removed, and cells were briefly washed with PBS once before introducing a culture media (DMEM, 10% FBS, 1 % Penicillinstreptomycin) with or without treatment (CB-100 : 52.8ug / mL, nidogen2 : 5ug / mL, insulin : 10nM, S961 : 200nM) and incubated for 24 hours at 37°C with 5% CO2. After 24 hours, culture media was collected. Pre and post incubated culture media were diluted 1000 times with water and glucose concentration of the media was measured with high sensitive glucose assay (Abeam) and glucose consumption was calculated.

[0204] Data Analysis and Statistics

[0205] Data were analyzed with R studio, Microsoft Excel, Image J, MATLAB, and GraphPad Prism. Calcium images were analyzed with MATLAB as described (Ng et al., Diabetes, 2022, 71 , pp 2384-2394). The statistical significance of each data is reported as mean ± SEM. Statistical significance was calculated as described in each figure legend.

[0206] INCORPORATION BY REFERENCE

[0207] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated as being incorporated by reference herein, including, for example U.S. Provisional Patent Application No. 63 / 661 ,573, filed June 19, 2024.

Claims

CLAIMSWhat is claimed is:1 . A method of treating type 1 diabetes (T1 D) by administering a therapeutically effective amount of a brown adipose tissue (BAT)- secreted factor to a subject in need thereof.

2. The method of claim 1 , wherein the BAT-secreted factor comprises a protein or fragment thereof from a BAT conditioned buffer exceeding a molecular weight of 10OkDa.

3. The method of claim 1 , wherein the BAT-secreted factor comprises a protein or fragment thereof from a BAT conditioned buffer with a molecular weight between 100kDa and 3kDa.

4. The method of claim 1 , wherein the BAT-secreted factor comprises a protein or fragment thereof from a BAT conditioned buffer with a molecular weight between 3kDa and 1 kDa.

5. The method of claim 1 , wherein the BAT-secreted factor comprises a protein or fragment thereof from a BAT conditioned buffer with a molecular weight smaller than 1 kDa.

6. The method of claim 1 , wherein the BAT-secreted factor comprises a protein or fragment thereof from a BAT conditioned buffer that is between 100 and 1 ,000 amino acids.

7. The method of claim 6, wherein the BAT-secreted factor comprises a protein or fragment thereof from a BAT conditioned buffer that is between 200 and 800 amino acids.

8. The method of claim 6, wherein the BAT-secreted factor comprises a protein or fragment thereof from a BAT conditioned buffer that is between 250 and 500 amino acids.

9. The method of any one of claims 2-8 wherein the protein or fragment thereof comprises nidogen-2 or a fragment thereof.

10. The method of any one of claims 1 -9, wherein administration of the BAT- secreted factor treats T1 D through improving glucose tolerance, improving insulin sensitivity, and / or promoting adipocyte differentiation.

11. A method of isolating a brown adipose tissue (BAT)-secreted factor for treating T1 D comprising: a. providing BAT cells; b. providing media in contact with the BAT cells; c. incubating the media and the BAT cells for a sufficient amount of time for the BAT-secreted factor to be present in the media; and d. filtering the media by membrane filtration to collect a molecular weight-specific fraction.

12. The method of claim 11 , further comprising: a. running the molecular weight-specific fraction through an anion exchange chromatography column; b. running the anion exchange chromatography column fraction through a size exclusion chromatography column; and c. isolating of an active protein fraction by mass spectrometry.

13. The method of claim 12, wherein a protein from the active protein fraction comprises nidogen-2.

14. The method of any one of claims 8-12, wherein the molecular weightspecific fraction is a >100kDa fraction, a 100kDa to 3kDa fraction, a 3kDa to 1 kDa fraction, or a <1 kDa fraction.

15. The method of claim 14, wherein the molecular weight-specific fraction is a >100kDa fraction.

Images