Carolinas Research Day 2021

EFFECTS OF ADIPOR1 AGONISTS ON INSULIN- INDEPENDENT GLUCOSE TRANSPORT PATHWAYS IN SKELETAL MUSCLE CELLS Joseph Metz, OMS-III; Marwan Mahmoud, OMS-II; Patrick D. Ellis Fisher, Ph.D Edward Via College of Osteopathic Medicine – Carolinas Campus; Gibbs Cancer Center, Spartanburg, SC Abstract Results Conclusions

Abstract # BIOM-6

Adiponectin is a protein-derived hormone that is produced in and secreted from adipocytes. Interestingly, adiponectin circulates in either trimeric, hexameric, or high-molecular weight conformations, with maturation being controlled by many chaperone proteins (4). Adiponectin is composed of 4 domains: a secretory signal sequence at the amino-terminus, a variable region, a collagenous-like domain, and a globular domain on its carboxyl end. These domains seem to play critical roles in the affinity of adiponectin at its subtype receptors AdipoR1 and AdipoR2. AdipoR1 is primarily expressed on skeletal myocyte membranes whereas AdipoR2 is primarily expressed on hepatocyte membranes. The globular carboxyl end domain seems to most strongly bind and activate AdipoR1, the receptor which has been demonstrated to be of more importance in glucose uptake (5). The perceived pathway upon binding to AdipoR1 is through activation of APPL1 (4). Propagation of the signal continues with activation of AMPK, p38 MAPK, Rab5, and IRS1/2 complex of the insulin receptor. p38 MAPK and Rab5 then allow an increase in GLUT4 translocation and expression on the cell membrane, allowing increased glucose uptake. GLUT4 translocation has been shown to increase in rat skeletal muscle when treated with globular adiponectin (gAd; 5). Activation of the IRS1/2 complex within insulin’s singling cascade increases activation of PI3K which allows glycogen and protein synthesis along with a further increase in GLUT4 translocation via PIP3 upon insulin binding (4). It is also believed that activation of AdipoR1 induces ceramidase activity, thus degrading ceramide into sphingosine and free fatty acids (6). Increased ceramide concentrations are believed to inhibit insulin intracellular signaling and induce insulin resistance (4,6,7). By degrading ceramide, insulin sensitivity could further be restored. The important role of adiponectin in glucose utilization demonstrates the utility of finding other compounds that agonize the AdipoR1 receptor. Through fluorescence polarization experiments, Sun et al (2013) demonstrated that matairesinol, a plant lignan, demonstrated agonistic characteristics towards AdipoR1. Lignans are polyphenolic compounds found in dietary fiber from plant based products. Furthermore, once ingested, matairesinol is metabolized into enterolactone and enterodiol by intestinal bacteria. Interestingly, the parent molecule and its metabolites have been reported to have biological activities such as decreasing free oxygen radicals, insulin resistance, and lipid peroxidation (2,8). These biological effects could possibly be explained from the activation of Adiponectin R1 and R2 receptors. As of 2015, the CDC estimates that nearly 30.3 million Americans were living with Diabetes mellitus and another 84.1 million Americans were classified as pre-diabetic (1). With such alarming up-trends, further elucidation of insulin- independent pathways for glucose metabolism is critical. Adiponectin is a protein-derived hormone that is produced and secreted from adipocytes and appears to help regulate glucose metabolism. A systematic review demonstrated that higher levels of circulating adiponectin are associated with a lower risk of developing type 2 diabetes and levels were inversely related to insulin resistance (2,3). Developing adiponectin receptor agonists could be an important turning point in the treatment of type 2 diabetes. We examined the effects of treating cultured human skeletal muscle cells (myocytes and myotubes) with the plant lignan matairesinol. Previous work has shown the binding and activation of the adiponectin receptor 1 (AdipoR1) by matairesinol (2). We hypothesized that matairesinol, along with its metabolites, can stimulate the AdipoR1 receptor and induce the translocation of insulin-responsive glucose transporter type 4 (GLUT4) to the plasma membrane, resulting in increased glucose uptake and a potential restoration of insulin sensitivity via downstream pathways. In the present study, we show that matairesinol treatment induces increased glucose uptake in human skeletal muscle tubes, similar to insulin and adiponectin. We have grown primary skeletal myocytes and differentiated them into polynucleated myotubes. Treating these with insulin, adiponectin, or our test compound matairesinol, we examined glucose uptake in muscle cells using a luciferase-based glucose uptake assay. We are currently working to determine the mechanism of activation, hypothesized to be via the AMP-activated Protein Kinase (AMPK) signaling pathway. Myocyte Cell Culture and Differentiation: Primary human myocytes were obtained from Cook MyoSite (SKMDC-1111, Lot 14F, 30F, and 8dayF) and ATCC (SkMC PCS-950- 010, Lot 70032406) and grown per manufacturers’ instructions. Differentiation into myotubes was induced by serum starvation using vendors’ proprietary differentiation media. Differentiation typically occurred between 3 and 5 days post-induction, but lengthened with higher passage numbers, concomitant with a reduction in differentiation efficiency. Glucose Uptake Analysis via luminescence: We utilized an assay to indirectly measure glucose uptake by recording luminescence after treatment with a special reagent (Promega Glucose Uptake-Glo). 2-deoxyglucose (2DG) was used in these assays as a glucose analog. When GLUT4 is stimulated to translocate to the cell membrane, 2DG transport into the cell increases. 2DG is phosphorylated to 2DG6P, thus trapping the sugar analog into the cell. A stop buffer was then added to the assay to stop 2DG transport, lyse cells, and inactivate proteins. 2DG6P Detection reagent was added into the cells and after a series of redox reactions, proluciferin is converted to luciferin which produces light signals relative to amount of 2DG uptake. Cells were grown in 96-well plates, differentiated, treated with 5 µg/mL gAd, 2 µM matairesinol, 1 µM insulin (positive control), or vehicle (0.1% DMSO) for 30 min at 37°C before being subjected to the assay. Globular Adiponectin Plasmid Construct, Expression, and Purification: To produce our own globular adiponectin (gAd), we designed a pET28a-derived bacterial plasmid containing an N-terminal His-tag, maltose-binding protein, TEV cleavage site, and codon-optimized gAd sequence. The construct was expressed in inclusion bodies and purified under denaturing conditions. Introduction Methods

The four different cell lines we used showed varying degrees of successful differentiation from myoblasts to mature myocytes. The cell lineage that gave us the highest yield of mature myocytes and phenotypically differentiated cells was the ATCC SkMC lineage. With the exception of the glucose uptake assay conducted on 2021-01-29 using Cook 8df, the rest of our data demonstrated increase glucose uptake with myotubes treated with matairesinol compared to vehicle. We were specifically interested in whether glucose was taken up by the cells, so the absolute concentration of glucose uptake by the myocytes is less relevant to our studies. However, it is important to note the day to day variability in our results. This is most likely due to differences in cell density and degree of differentiation as well as day to day variability of the assay itself, which implies that repeated studies are important to further validate this data and to decrease any error. It’s important to mention that another reason that the degree of glucose uptake is less relevant than whether there was a glucose uptake response is because the cell lines are all genotypically different. Therefore, it is plausible that certain cell lines are more susceptible to glucose uptake stimulated by gAd or matairesinol compared to insulin. Overall, our data support that matairesinol does increase glucose uptake compared to vehicle. We successfully demonstrated adequate plasmid uptake by E. coli. The bacteria cells then effectively produced our protein of interest (gAd). We also demonstrated that 8M urea solubilized the inclusion bodies that were produced. Lastly we demonstrated that our Ni 2+ chromatography column with increasing imidazole concentration successfully purified our recombinant gAd.

20 µm

Undifferentiated

Differentiating

Figure 1. Myocyte Cell Differentiation (ATCC SkMc lineage) Representative images of successful differentiation of myoblast to mature polynuclear myotubes (red arrow). Undifferentiated cells (passage 3; left) and differentiating cells (passage 4, 8 days post serum starvation; right). Phase contrast, scale bar 20 µm.

Future Directions

Our next steps on this project are to further elucidate the downstream signaling cascade of AdipoR1 through Western Blotting and qPCR. We would also like to investigate the binding ability of the two metabolites of matairesinol, enterodiol and enterolactone, to AdipoR1 and effects on glucose uptake. We will also test structure and function of our recombinant gAd via glucose uptake assays. Lastly, we would like to elucidate the roles that these agonists play in regulating AdipoR1 and AdipoR2 ceramidase activity and its potential role in fuel metabolism. Clinically, these studies could lay a foundation for future work creating small molecule compounds or agonist monoclonal or bivalent antibodies that could be used to decrease the use of insulin in type 2 diabetic patients. Our results could inspire new first-line medications for diabetic patients, due to the potential cardiovascular benefits associated with AdipoR1 activation and mechanistically solving the pathophysiological problem in DM2T, insulin insensitivity.

References

Figure 2. Glucose uptake analysis for 4 myoblast cell lines. Bar graphs demonstrating the fold change of glucose uptake as compared to vehicle (dotted line at Y=1). Replicate values shown, columns and numbers show mean, error bars show S.D. Cook 8dayF: experiment date 2021-01-29 passage 8 (P8) day 4 post-differentiation (d4), N=1; 2021-02-15 P9 d6, N=2. Cook 30F 2021-01-29 P9 d4, N=3 (N=2 for gAd); 2021-02-15 P8 d6, N=3 (N=2 for matairesinol). Cook 14F 2020-12-22 P4 d3, N=3. ATCC SkMC 2021-02-09 P9 d7, N=3.

1. CDC Division of Diabetes Translation. National Diabetes Statistics Report. 2017. https://www.cdc.gov/diabetes/pdfs/data/statistics/national-diabetes-statistics-report.pdf 2. Sun Y, Zang Z, Zhong L, et al. Identification of adiponectin receptor agonist utilizing a fluorescence polarization based high throughput assay. PLoS ONE. 2013;8(5):e63354 3. Shanshan L, Shin HJ, Ding EL, et al. Adiponectin levels and risk of type 2 diabetes: a systematic review and meta-analysis. JAMA. 2009;302(2):179-188 4. Achari A and Jain S. Adiponectin. A Therapeutic Target for Obesity, Diabetes, and Endothelial Dysfunctions. Int. J. Mol. Sci. 2017;18(6):1321 5. Ceddia RB, Somwar R, Maida A, et al. Globular adiponectin increases GLUT4 translocation and glucose uptake but reduces glycogen synthesis in rat skeletal muscle. Diabetologia. 2005;48:132-139. 6. Holland WL, Brozinick JT, Wang LP, et al. Inhibition of ceramide synthesis ameliorates glucocorticoid-, saturated-fat-, and obesity-induced insulin resistance. Cell Metab . 2007;5:167-179 7. Sharma A and Holland W. Adiponectin and its hydrolase-activated receptors . JNSCI. 2017;3(6) 8. Milder I, Freskens E, Arts I, et al. Intake of the plant lignans secoisolariciresinol, matairesinol, lariciresinol and pinoresinol in dutch men and women. The Journal of Nutrition . 2005;135(5):1202-1207

A.

B.

C.

Acknowledgements

Figure 3 . Purification of globular adiponectin. gAd plasmid construct map ( A ). Important features worth noting include: 1. The N- terminal 6xHis tag for purification with affinity chromatography. 2. Maltose-Binding Protein (MBP) tag to allow greater solubility and expression. 3. Tobacco Etch Virus (TEV) protease cleavage site for tag removal. 4. codon-optimized gAd gene (gAcrp30). 5. Kanamycin A resistance to allow growth in kanamycin. 6. Lac Operon allowing for expression under specific conditions. Gel image of the expression of gAd (60.1 kDa) and the eluted protein fractions from the Ni 2+ column ( B ). Of note, 8M urea was used to solubilize inclusion bodies and the band at 38 kDa is an undefined impurity. Representative image of the UV absorbance trace during affinity chromatography washing and elution during purification ( C ). The elution fractions from the column indicate an increase in the imidazole concentration allowing for purification.

We would like to thank the VCOM Research Eureka Accelerator Program (REAP) for graciously funding this project. We would also like to thank AddGene for the providing the MBP-TEV plasmid and GeneWiz for synthesizing and inserting a codon-optimized gAd gene into the plasmid construct.

15

2 0 2 1 R e s e a r c h R e c o g n i t i o n D a y