DIABETES MANAGEMENT PRESENTER: DR. SOPHIA BRYAN 1 SRC NOVEMBER, - - PowerPoint PPT Presentation

THE ROLE OF NITRIC OXIDE SYNTHASE INHIBITORS IN

DIABETES MANAGEMENT

PRESENTER: DR. SOPHIA BRYAN

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SRC NOVEMBER, 2018

INTRODUCTION

 Nitric oxide (NO) is a simple,

colourless gas.

 Formed naturally from electric

charges during lightning.

 Emitted into the atmosphere from

cigarette smoke, acid rain, automobiles and as a by-product of the combustion of fuels that causes environmental pollution.

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“Molecule of the Year” in 1992.

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INTRODUCTION

BIOSYNTHESIS OF NITRIC OXIDE:

NOS (nNOS, eNOS and iNOS) O2, NADPH

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NO DONORS: S-NITROSOTHIOLS

S-nitrosothiols (RSNOs) are found in the body

and others can be synthetically made. The S-nitrosothiols used in this research were:

• S-nitrosocaptopril (CapSNO)
• S-nitroso-N-acetyl-D,L-penicillamine (SNAP)
• S-nitrosoglutathione (GSNO)
• CapSNO and SNAP are synthetically made and

GSNO occurs naturally.

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NOS INHIBITORS

The two NOS inhibitors used were:

• NG-monomethyl-L-arginine (L-NMMA)
• NG-nitro-L-arginine methyl ester (L-NAME)
• L-NMMA is found naturally in the body and

L-NAME is synthetically made.

• Used in studies to decrease NO bioavailability
• r to establish the physiological role of NO.

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NITRIC OXIDE (NO)

Plays a number of important roles in the body

and is well known as a vasodilator.

However, it is also implicated in a number of

pathological conditions in which NO is either under or overproduced. This leads to disease conditions such as sepsis, atherosclerosis, hypertension and diabetes.

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DIABETES MELLITUS (DM)

 In 2017, approx. 425 million persons had diabetes

worldwide and this is expected to rise to 629 million by the year 2045. (IDF, 2017)

Globally, 1.6 million deaths were directly caused by

diabetes in 2016. (WHO, 2016)

In Jamaica, DM is one of the leading causes of

death (WHO, 2018)

Billions of US dollars is spent on healthcare for

diabetics in many countries. (IDF, 2017)

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DIABETES MELLITUS

Diabetes mellitus (DM) is a chronic health condition where the body is either unable to produce enough insulin or use insulin properly to control the amount of glucose in the blood, leading to elevated levels in the blood stream. Three main types of DM:

Type 1 Type 2 Gestational Diabetes

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DIABETES MELLITUS

Type 1: An autoimmune disease, where the body’s immune system destroys the beta cells of the

• pancreas. Affects ≈10% of DM patients.

Type 2: Characterized usually by insulin resistance. The pancreas produces insulin, but either this is not sufficient or the body is unable to utilize it effectively (insulin resistance). Affects ≈ 90% of diabetics. Gestational diabetes: Develops in some women late in pregnancy due to hormonal changes in pregnancy. Usually resolved after delivery.

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NORMAL BLOOD GLUCOSE REGULATION

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OBJECTIVES

To determine the effects of the RSNOs

(CapSNO, SNAP & GSNO) and the NOS inhibitors, L-NAME and L-NMMA on blood glucose, insulin and glucagon concentration.

To compare the pancreas of rats treated with

CapSNO, SNAP & GSNO with L-NAME & L-NMMA treated rats for histology studies.

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METHOD

Rats were divided into 2 groups (6 per group):

• normal untreated (control)
• normal treated (samples)
• Control (0.9% saline)
• RSNOs - 12.5 mg/kg body weight (BW) via

intravenous (i.v) administration.

• NOS inhibitors - 30 mg/kg BW (i.v admin.)

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BLOOD GLUCOSE CONCENTRATION

• Modified version of the Oral Glucose Tolerance

Test (OGTT) was used (Balon et al., 1999)

• Readings were obtained using the Accu-Chek

Advantage glucometer.

• Rats were fasted overnight (12 hours)
• Glucose load (1.75 g/kg BW) was administered
• rally.

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METHODS CONT’D

HORMONE ANALYSIS:

Commercially available ELISA kits for serum insulin (Mercodia, USA) and glucagon concentration (ALPCO, USA) were purchased.

HISTOLOGY STUDIES: Under the supervision of

an experienced Anatomical Pathologist.

Rat pancreas was excised and fixed in Bouin’s solution. Slides

were prepared and viewed under a light microscope to assess pancreas integrity.

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RESULTS: GLUCOSE METABOLISM

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FIGURE 1: Effect of CapSNO vs L-NAME & L-NMMA on Blood Glucose Concentration

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2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 30 60 90 120 150 180 210

Blood Glucose Concentration (mM) Time (min) CapSNO L-NAME L-NMMA Saline (Control)

* * * * * * * *

* P<0.05

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2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 30 60 90 120 150 180 210

Blood Glucose Concentration (mM) Time (min) SNAP L-NAME L-NMMA Saline (Control) * P<0.05

FIGURE 2: Effect of SNAP vs L-NAME &

L-NMMA on Blood Glucose Concentration * * * * * *

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FIGURE 3: Effect of GSNO vs L-NAME &

L-NMMA on Blood Glucose Concentration

2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

30 60 90 120 150 180 210

Blood Glucose Concentration (mM) Time (min) GSNO L-NAME L-NMMA Saline (Control) * P<0.05

* * * * *

HORMONE ANALYSIS: INSULIN & GLUCAGON

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FIGURE 4: Effect of the RSNOs vs Inhibitors

• n Glucagon Concentration

50.0 70.0 90.0 110.0 130.0 150.0 170.0 190.0 210.0 230.0

Glucagon Concentration (pg/mL) Time (90 min interval)

CapSNO SNAP GSNO L-NAME L-NMMA Saline (control)

* P<0.05

* * *

0.2 0.4 0.6 0.8 1 1.2

Insulin Concentration (μg/L) Time (90 min interval) CapSNO SNAP GSNO L-NAME L-NMMA Saline (control)

* P<0.05

FIGURE 5: Effects of RSNOs vs Inhibitors on

Insulin Concentration * * *

HISTOLOGICAL STUDIES

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THE PANCREAS

Consists of two portions:

• endocrine
• exocrine
• Plays a dual role in the body.
• Exocrine portion secretes pancreatic juices which

contains digestive enzymes that help in the breakdown of foods and the absorption of nutrients.

• The islet of Langerhans which makes up the

endocrine portion secretes the hormones insulin and glucagon. Four types of cells are found here- alpha, beta, delta and gamma.

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Untreated Rat Pancreas (Control)

berry-like clusters of cells called acini

Islet of Langerhans

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B. Rats treated with L-NAME & L-NMMA. Heavily dense with beta (β)-cells which were stained purple. Mag x100 A. Rats treated with CapSNO, SNAP & GSNO. Only alpha (α)-cells which were stained yellow were observed. Mag x400 (oil immersion)

Islet of Langerhans in the Rat Pancreas

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Wave of new β-cells being generated. Precursors to pancreatic islets. Mag x400 Wave of new β- cells was seen around some arteries. Mag x400

Rat Pancreas: L-NAME and L-NMMA TREATMENT

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RAT PANCREAS: L-NAME AND L-NMMA TREATMENT

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Proliferation

• f new ducts.

(Mag x400) Dense area of β-cells surrounding new ducts.

CONCLUSION

RSNOs: CapSNO, GSNO & SNAP (12.5 mg/kg BW)

caused hyperglycaemia (elevated blood glucose levels).

Inhibitors: L-NAME and L-NMMA (30 mg/kg BW)

significantly lowered the postprandial blood glucose concentration.

Novel findings from this research, were that treatment

with L-NAME and L-NMMA stimulated the generation

• f new beta-cells and also the proliferation of ducts

which have the potential to differentiate into new insulin-secreting beta cells (new source of beta-cells).

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CONCLUSION

Bench to bedside: Opportunities for wealth creation

through the development of new drug formulations to treat DM.

Pancreas transplantation. Beta-cell regeneration may

be a potential innovative therapy that someday may even reverse diabetes.

Will result in a better quality of life for millions of

persons affected with DM worldwide, including Jamaica and help to reduce the high healthcare costs associated with DM.

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REFERENCES

 International Diabetes Federation (IDF), IDF Diabetes Atlas Eighth Edition, 2017.

https://idf.org/52-about-diabetes.html

 World Health Organization (WHO), Global Report 2016.

http://www.who.int/diabetes/en/

 World Health Organization (WHO), Non-communicable diseases (NCD) Country

Profile 2018. http://www.who.int/nmh/countries/jam_en.pdf

 Bouwens, L., Wang, R. N., De Blay, E., Pipeleers, D. G., & Kloppel, G. (1994).

Cytokeratins as markers of ductal cell differentiation and islet neogenesis in the neotal rat pancreas. Diabetes 43, 1279-1283.

 Bryan, S., Alexander-Lindo, R., Dasgupta, T., & McGrowder, D. (2011a). The Effect

• f Nitric Oxide Inhibitors and S-Nitrosothiols on Hemodynamic Parameters in an

Animal model. Open Access Animal Physiology, 3, 1-8.

 Bryan, S., Alexander-Lindo, R., Dasgupta, T., & McGrowder, D. (2011b). The Effect

• f Nitric Oxide Inhibitors and S-nitroso-N-acetylpenicillamine on Glucose

Concentration in an Animal Model. J Nat Sci Biol Med, 2(1), 80-86. doi: 10.4103/0976-9668.82314

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Thank You!