Diabetes is the result of high circulating glucose levels due to the inability to effectively remove glucose from the blood stream. It is a growing epidemic in the United States and abroad. Type II, most common form of diabetes, have normal insulin secretion in response to glucose, but have a lower sensitivity to insulin. The insulin receptor, which is a ligand-activated receptor and a member of the receptor tyrosine kinase (RTK) family, plays a vital role in regulatory functions in growth, cell differentiation and metabolism (Lee and Pilch, 1994). This receptor is a dimer composed of an alpha and beta subunits. Upon binding of insulin, the insulin receptor autophosphorylates. The exact mechanism in unknown but a signal cascade is produced and in skeletal muscles cells, a highly efficient glucose transporter (GLUT-4) travels to the cell surface.

Recent work has shown chromium lowers circulating blood glucose levels. Chromium seems to increased insulin receptor activity but mechanism in which this occurs has not been fully understood. Cromium oligopeptide has been shown to bind to insulin activated insulin receptor and lower glucose and insulin levels in diabetic patients (Cefalu, 2002). Chromium picolinate supplements have shown to increase insulin sensitivity and Glut-4 translocation in obese rats (Cefalu, 2002). Creatine monohydrate has also been recently found that they can have an lower on glucose levels without changes in insulin levels (Earnest, 1996).

While Creatine has been shown to decrease glucose levels without altering insulin, the mechanism by which this occurs has not been fully elucidated. Therefore, this study examined the effect creatine had on insulin receptor levels (97 kDa) and phosphorylation status. Then as a measure of activity, we examined the effect of creatine on expression of insulin regulated gene phosphoenolpyruvate carboxykinase (PEPCK). PEPCK is a rate controlling gene in glucogenesis which is dominantly inhibited by insulin and plays an important role in glucose homeostasis (Lee and Pilch, 1994).

Material and MethodsH4IIE Cell Culture:

H4IIE rat hepatoma cells were brought up maintained in alpha essential MEM with 12% horse and bovine serum with 1% P/S. The cells were split 1:10 at 80% confluence.

Protein Treatments and Isolation:

Night before treatments, cells were shifted into incomplete media. Cells were treated with 500 uM creatine alone at 16 hr and 24 hr prior to isolation. Creatine (500 uM) and insulin treatments (50 nM, 5 min. prior to isolation) were performed at times 2hr, 12 hr, 16 hr and 24 hr after creatine treatment. Cells were washed with PBS and lysated using RIPA lysis buffer with protease inhibitors NaF (50 mM) and Sodium Phosphosphate (30mM). Protein quantification was performed with a BCA assay follow manufactures protocol (Pierce).

RNA Treatments and Isolation:

Cells were plated in 6 well plates (350,000 cells/well) and allowed to attach overnight. Total cellular RNA were obtained using Trizol reagent kit per manufacture instructions (Gibco-BRL ). H4IIE cells were treated with 10 nm insulin treatment performed 2 hr prior to Dex and 500 um creatine treatment was performed 24 hr prior to isolation in various treatments. DNASE treatment was performed and RNA concentrations were quantified (260nm wavelength).

RT-PCR:

Total RNA (0.5 ug) was then converted to cDNA and amplified using QuantumRNA kits (Ambion). Reverse transcriptase reaction was run for 60 minutes at 42°C. For the PEPCK PCR reaction, 1.5 µl of the RT reaction was loaded with 0.5 ul PEPCK upper primer(5’-GCATGGGGTGTTTGTAGGAG) and 0.5 ul PEPCK lower primers(5’-ACTTGCCGTTTTTGTCTTTC).   PEPCK PCR was run after a 5 minute incubation at 94 C, samples were amplified for 30 cycles (94° C for 30 sec, 52º C for 60 sec, 72° C for 40 sec), then reheated to 72° C for 5 min. and then cooled to 4° C. For 18S PCR 1.5 ul of RT reaction was loaded with 1 ul of universal 18S primers set. After a 5 minute incubation at 94° C, samples were amplified for 30 cycles (94° C for 30 sec, 57° C for 30 sec and 72° C for 30 sec), then heated to 72° C for 5 minutes and then cooled to 4° C.

Fig 2. RT-PCR gel electrophoresis with PEPCK upper and lower primer and 18S primers (A+B): Expression of PEPCK and 18S in H4IIE cells exposed to creatine(cre), insulin(ins), and dexmethionine. PEPCK primers produce added produced 222 bp size fragment while

universal 18S primers produced a 353 bp fragment. Gels (1.5% agarose) were run at 100 v for 30 minutes.

Data

Results

DiscussionIn H4IIE rat hepatoma cells, according to our data, creatine

does not alter insulin receptor pathway. Creatine does not appear to increase IR phosphorylation or total insulin receptor protein levels. Creatine did not alter PEPCK gene expression, a known insulin sensitive gene.

Creatine treatment did appear to increase phosphotyrosine band (125 kDa), which could be the result of increased phosphorylation of focal adhesion kinase (FAK). FAK has been recently found to involved in insulin-mimetic signaling (Mueller, 2000). FAK has been also found to regulate insulin-stimulated glycogen production (Huang, 2002). This leads to the possibility that creatine stimulates FAK via non-insulin receptor pathway.

The lower band that appeared on the IR blot at treatments at 2, 12 and 16 hr with insulin + creatine could be possible degradation products of the insulin receptor. The insulin receptor is degraded to 88 kDa form before it is finally degraded to a 50 kDa form. (Kathuria,1986). This occurred when insulin is internalized upon binding of insulin and slowly degraded (Trishitta, 1989). This lower smaller degradation product (88 kDa) was similar to the band sizes found in creatine + insulin treatments. Creatine may cause internalization of IR which becomes down regulated when treated with insulin + creatine, which according to our data does not occur in treatments of creatine alone.

Creatine does not alter total insulin receptor levels or insulin receptor phosphorlation. There was increased phosphorlation at 125 kDa which could be result of FAK. This leads to the possibility that creatine stimulates a non-insulin receptor mediated pathway, which will be the focus of future research.

Literature Cited

Cefalu, W.T., Wang, Z. Q., Zhang, X. H., Baldor, L. C. and J. C. Russell. 2002. Oral Chromium Picolinate Improves Carbohydrate and Lipid Metabolism and Enhances Skeletal Muscle Glut-4 Translocation in Obese Hyper-insulinemic (JCR-LA Corpulent) Rats. Journal of Nutrition. 132:1107-1114.

Davis, M.C., and J.B. Vincent. 1997. Chromium Oligopeptide Activates Insulin Receptor Tyrosine Kinase Activity. Biochemistry 36: 4382-4385.

Earnest, C.P., Almada, A.L., and T.L. Mitchell. 1996. High-performance Capillary Electrophoresis pure Creatine Monohydrate Reduces Blood Lipids in Men and Women. Clinical Science 91: 113-118.

Huang, D., Cheung, A. T., Parsons, J. T., and M. Bryer-Ash. 2002. Focal Adhesion Kinase (FAK) Regulates Insulin-stimulated Glycogen Synthesis in Hepatocytes. Journal of Biological Chemistry. Vol. 277. Issue 20: 18151-18160.

Kathuria, S., Hartman, S., Grunfeld, C., Ramachandran, J., and Y. Fujita-Yamguchi. 1986. Differential Sensitivity of Two Functions of the Insulin Receptor to the Associated Proteolysis: Kinase Action and Hormone Binding. Biochemistry. Vol. 83, 8570-8574.

Lee, J., and P. F. Pilch. 1994. The insulin receptor: structure, function, and signaling. American Journal of Physiology. 266(Cell Physiology 35) C319-C334.

Mueller, G., Wied, S., and W. Frick. 2000. Cross Talk of pp125FAK and pp59Lyn Non-Receptor Tyrosine Kinases to Insulin-Mimetic Signaling in Adipocytes. Molecular and Cellular Biology . Vol. 20. Issue 13; 4708-4723.

Trischitta, V., Wong, K.Y., Burnetti, A., Scalisi, R., Vigner, R. and I.D. Goldfine. 1989. Endocytosis, Recycling, and Degradation of Insulin Receptor Studies with Monoclonal Anti-receptor Antibodies That Do Not Activated Receptor Kinase. Journal of Biological Chemistry. Vol 264, No. 9;5041-5046.

Effect of Creatine on Total Insulin Receptor Levels, Phosphorylation and PEPCK gene expression

Joshua Burton

Department of Biological Sciences, York College of Pennsylvania

Introduction

Fig 1. Total Insulin Receptor Protein Level and Phosphotrosine Levels: Poly-Acrylamide gels were loaded with 40 ug of protein samples and run at 170v, 500 mA for 60 minutes. Proteins bands were transferred onto nitrocellulose membrane for 1 hr at 100v and 350 mA. Insulin Receptor was detected by treating the nitrocellulose membrane overnight in TBS with anti-insulin receptor, b-subunit antibody made in rabbit. Phosphotyrosine band was detected by treating the nitrocellulose membrane overnight in TBS with PY99 antibody made from mouse. Primary antibodies were incubated at 4 degrees C overnight and secondary antibody conjugated with Color sensitive horseradish peroxidase linked to secondary antibody was used conjugate and precipitate to primary antibodies for 30 minutes.

MW Insulin Creatine +Insulin Creatine Control24hr 16hr 12 hr 2hr

24hr 16hr SF SKDalt.

203

126

80

IR*

IR*

Creatine + Insulin Creatine Control

PTYR

Insulin

Insulin+Creatine24hr 16hr 12hr

2hr

Creatine24hr 16hr

MW

ControlSF S KDal

t.

203

80

126

PTYR

126

PEPCK

I/D I/C D I C+I/D C C/D CON 100 bp MWI/D I/C D I C+I/D C C/D CON 100 bp MW

18S

PEPCK

18S

A

B

C+I/D I C C/D I/C I/D D CON 100 bp MWC+I/D I C C/D I/C I/D D CON 100 bp MW

PEPCK

18S

B

A B

IRS-1

Ras PI-3-K

SignalGLUT 4

Insulin receptor

CreatineInsulin

Glucose

Alter Gene Expression

Fig 3. Proposed mechanism of creatine induced phosphorylation of the insulin receptor. 

Creatine increases insulin-induced autophosphorylation of IR.  This results in activation of signaling molecules resulting in translocation of the Glut4 to the cell membrane increasing glucose uptake (Adapted from Lee and Pilch, 1994).  Abbreviations: Insulin Receptor (IR), Insulin Receptor Substrate (IRS)-1, Phosphatidylinositol 3-hydroxy Kinase (PI3K), and Glucose Transporter (Glut4).

Ins/Dex Ins/Cre Dex Ins Cre+Ins/Dex Cre Cre/Dex Con Cre+Ins/Dex Ins Cre Cre/Dex Ins/Cre Ins/Dex Dex Con

Acknowledgments: Special thanks to Dr. Ronald Kaltreider for his guidance and patience.

kDa MW Ins 24 hr 16 hr 12 hr 2 hr 24 hr 16 hr SF S kDa Ins 24 hr 16 hr 12hr 2hr 24 hr 16 hr MW SF S

Creatine + Insulin Creatine Control

• With creatine alone, no gross changes were observed. Total IR levels (97 kDa) was not present at 2, 12, 16, and 24 hr creatine + insulin treatments. A smaller protein band did appear at 2, 12 and 16 hr treatments with creatine and insulin.

•With creatine alone, no gross changes in phosphotyrosine activity was observed. Phosphotyrosine activity was present at 2 hr creatine + insulin treatments but disappeared at 12, 16, and 24 hr treatments with creatine + insulin.

• Increased phosphotyrosine band (125 kDa) was observed in treatments with creatine alone 16 and 24 hr and in 2 hr creatine + insulin treatment

• Dex treatments caused a slightly higher PEPCK gene expression, while insulin decreased expression. Creatine treatments alone or in combination did not effect PEPCK gene expression.

203

80

203

126

80