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

T HE ROLE OF N ITRIC O XIDE S YNTHASE I NHIBITORS IN DIABETES MANAGEMENT PRESENTER: DR. SOPHIA BRYAN 1 SRC NOVEMBER, 2018

I NTRODUCTION  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 2 environmental pollution.

I NTRODUCTION  “Molecule of the Year” in 1992. 3

B IOSYNTHESIS OF N ITRIC OXIDE : NOS (nNOS, eNOS and iNOS) O 2 , NADPH 4

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: o S-nitrosocaptopril (CapSNO) o S-nitroso-N-acetyl-D,L-penicillamine (SNAP) o S-nitrosoglutathione (GSNO)  CapSNO and SNAP are synthetically made and 5 GSNO occurs naturally.

NOS INHIBITORS The two NOS inhibitors used were: o N G -monomethyl-L-arginine (L-NMMA) o N G- 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 or to establish the physiological role of NO. 6

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. 7

D IABETES 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 8 diabetics in many countries. (IDF, 2017)

D IABETES M ELLITUS 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 9  Gestational Diabetes

D IABETES M ELLITUS 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. 10 Usually resolved after delivery.

NORMAL BLOOD GLUCOSE REGULATION 11

O BJECTIVES  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. 12

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.) 13

B LOOD G LUCOSE C ONCENTRATION o Modified version of the Oral Glucose Tolerance Test (OGTT) was used (Balon et al., 1999) o Readings were obtained using the Accu-Chek Advantage glucometer. o Rats were fasted overnight (12 hours) o Glucose load (1.75 g/kg BW) was administered 14 orally.

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. 15

RESULTS: GLUCOSE METABOLISM 16

FIGURE 1: Effect of CapSNO vs L-NAME & L-NMMA on Blood Glucose Concentration CapSNO L-NAME L-NMMA Saline (Control) 7.50 Blood Glucose Concentration (mM) 7.00 * 6.50 6.00 5.50 * * 5.00 * 4.50 * * * 4.00 * 3.50 3.00 2.50 0 30 60 90 120 150 180 210 Time (min) 17 * P<0.05

FIGURE 2: Effect of SNAP vs L-NAME & L-NMMA on Blood Glucose Concentration SNAP L-NAME L-NMMA Saline (Control) 7.50 Blood Glucose Concentration (mM) 7.00 6.50 * * * 6.00 5.50 5.00 * 4.50 * 4.00 * 3.50 3.00 2.50 0 30 60 90 120 150 180 210 Time (min) * P<0.05 18

FIGURE 3: Effect of GSNO vs L-NAME & L-NMMA on Blood Glucose Concentration GSNO L-NAME L-NMMA Saline (Control) 7.50 Blood Glucose Concentration (mM) 7.00 6.50 * 6.00 5.50 * 5.00 * 4.50 * 4.00 * 3.50 3.00 2.50 0 30 60 90 120 150 180 210 19 * P<0.05 Time (min)

HORMONE ANALYSIS: INSULIN & GLUCAGON 20

FIGURE 4: Effect of the RSNOs vs Inhibitors on Glucagon Concentration CapSNO SNAP GSNO L-NAME L-NMMA Saline (control) 230.0 Glucagon Concentration (pg/mL) 210.0 * 190.0 * 170.0 * 150.0 130.0 110.0 90.0 70.0 50.0 Time (90 min interval) * P<0.05

FIGURE 5: Effects of RSNOs vs Inhibitors on Insulin Concentration CapSNO SNAP GSNO L-NAME L-NMMA Saline (control) Insulin Concentration ( μ g/L) 1.2 1 0.8 0.6 * * 0.4 * 0.2 0 Time (90 min interval) * P<0.05

HISTOLOGICAL STUDIES 23

T HE P ANCREAS Consists of two portions: o endocrine o exocrine o Plays a dual role in the body. o Exocrine portion secretes pancreatic juices which contains digestive enzymes that help in the breakdown of foods and the absorption of nutrients. o The islet of Langerhans which makes up the endocrine portion secretes the hormones insulin 24 and glucagon. Four types of cells are found here- alpha, beta, delta and gamma.

Untreated Rat Pancreas (Control) berry-like clusters of cells called acini Islet of Langerhans 25

Islet of Langerhans in the Rat Pancreas A. Rats treated with CapSNO, SNAP & GSNO. Only alpha (α) -cells which were stained yellow were observed. Mag x400 (oil immersion) B. Rats treated with L-NAME & L-NMMA. Heavily dense with beta ( β )-cells which were stained purple. Mag x100 26

Rat Pancreas: L-NAME and L-NMMA TREATMENT 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 27

RAT PANCREAS : L - NAME AND L - NMMA TREATMENT Proliferation of new ducts. (Mag x400) Dense area of β -cells surrounding new ducts. 28

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 of new beta-cells and also the proliferation of ducts which have the potential to differentiate into new 29 insulin-secreting beta cells (new source of beta-cells).

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. 30

R EFERENCES  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., & M c Growder, D. (2011a). The Effect of 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 of Nitric Oxide Inhibitors and S-nitroso-N-acetylpenicillamine on Glucose 31 Concentration in an Animal Model. J Nat Sci Biol Med, 2 (1), 80-86. doi: 10.4103/0976-9668.82314

Thank You! 32