Molecular Mechanisms of Type 2 Diabetes Mellitus
Molecular Mechanisms of Type 2 Diabetes Mellitus
This project is designed to evaluate the molecular mechanisms involved in the early
 development of endothelial dysfunction in type 2 diabetic patients. The investigators intend
 to correlate increases in insulin signaling pathway activity following pioglitazone therapy
 with improvements in nitric oxide synthase expression in skeletal muscle. In addition, the
 investigators will evaluate vascular responses and in vivo nitric oxide release during
 administration of acetylcholine and nitroprusside in patients with type 2 diabetes. Enhanced
 knowledge of the molecular mechanisms responsible for endothelial dysfunction, an early
 abnormality in the pathogenesis of atherosclerosis, is critical before novel therapies to
 arrest or delay the appearance of cardiovascular complications in diabetes can be developed.

 The investigators intend to recruit fifty type 2 diabetic patients treated with diet alone or
 diet plus sulfonylureas or meglitinides and add Pioglitazone (45 mg), an insulin sensitizer,
 for 6 months. In addition to assessment of clinical and metabolic parameters, insulin
 sensitivity and brachial artery and skin microcirculatory responses to acetylcholine and
 nitroprusside in combination with simultaneous determination of nitric oxide release will be
 documented before, 3 and 6 months after Pioglitazone therapy is initiated. Circulating levels
 of markers of endothelial damage (VCAM, ICAM, selectins), inflammation (C-reactive protein
 and interleukins), increased coagulability (PAI-1) as well as lipids and apolipoproteins will
 measured during the study. Skeletal muscle biopsies will be performed during the euglycemic
 insulin clamp before and 6 months after therapy for measurements of NO synthase activity and
 key elements of the insulin signal transduction pathway involved in the regulation of glucose
 metabolism (IRS-1, PI-3 kinase, PI-3 kinase associated with IRS-1 and the mitogenesis
 MAP-kinase.

 Type 2 diabetes confers a substantial increase in the risk of cardiovascular disease. This is
 believed to be due, in part, to endothelial dysfunction, which correlates closely with
 impaired vascular responsiveness. Our study will clarify further the extent to which
 resistance to insulin action and impaired nitric oxide release from endothelial cells are
 interrelated. We also expect to demonstrate that insulin sensitizers (pioglitazone) can help
 to restore normal endothelial function, and ultimately prevent/delay the appearance of
 vascular disease in patients with type 2 diabetes.
Specific Aims

 To test the global hypothesis that increases in insulin signaling through either PI-3-kinase
 or MAP kinase pathways correlate with improvements in NO production or NOS expression in
 skeletal muscle following pioglitazone therapy, we will determine whether 6 months of
 Pioglitazone therapy:

 i. Increases the activities of muscle NO synthase, IRS-1, PI-3 kinase, PI3-kinase associated
 with IRS-1 and/or MAP-kinase ii. Improves brachial arterial endothelial function In addition,
 we will determine whether 6 months of pioglitazone therapy improves abnormal cardiovascular
 disease risk factors in type 2 diabetic patients by documenting the effects of the drug on
 plasma lipoprotein profile and on circulating levels of markers of hypercoagulability,
 inflammatory and endothelial damage.

 Experimental Plan Subjects. Adult type 2 diabetic individuals ranging from 18-65 yrs, with
 BMI between 24 and 37 kg/m2 and mean hemoglobin A1c levels equal to 6.5 and 9.0% will be
 studied. A total of 50 subjects will be recruited. There are over 8,000 type 2 diabetic
 patients under treatment at the TDI with a total of approximately 90,000 visits per year
 registered in the year 2000. As the main source of diabetes care and education to a
 population of predominantly Hispanic Americans, the TDI also is responsible for periodic
 screening in the community. Thus, we do not anticipate any problems in recruiting 50 subjects
 required for the present study. All patients will be normotensive, free of significant
 medical problems other than diabetes, and either on diet alone or diet plus sulfonylurea (or
 meglitinides) therapy. Subjects who have received or are receiving treatment with a
 thiazolidinedione (TZD) will be excluded. We previously have shown that sulfonyureas have no
 direct insulin sensitizing effect.

 Research Design. General Approach. An open-label prospective clinical trial including 50 type
 2 diabetic patients (25 diet-treated and 25 treated with diet plus sulfonylurea) will have
 pioglitazone, 45 mg daily; added to their therapeutic regimen. All patients will be closely
 monitored and, in addition to periodic contacts and clinical visits, metabolic and vascular
 parameters will be assessed at the beginning and after 3 and 6 months of therapy. Euglycemic
 hyperinsulinemic clamp with muscle biopsies will be performed at the beginning and after 6
 months of treatment.

 Subject Recruitment. Potential volunteers will be invited for a screening visit (Visit 1)
 during which subjects will be instructed in English (with Spanish translation when
 appropriate) about the nature of the study, eligibility criteria and required commitments, as
 well as the potential risks and benefits anticipated for each individual and for the
 community in general. Written informed consent for participation in the clinical trial from
 individuals thought to be eligible to participate will be obtained at Visit 1.

 At Visit 1 a complete medical history (including a list of current medications, reproductive
 status and menstrual history for females), physical examination (including height and
 weight), and vital signs will be obtained. Three consecutive blood pressure measurements will
 be performed in the sitting position using an automated arm cuff and only individuals with a
 blood pressure less than 140/90 mmHg will be considered eligible. A urinalysis will be
 performed and blood will be drawn after an overnight fast for measurement of complete blood
 count (hematocrit, white cells and platelets), plasma glucose, free T4 and TSH; SMA 20, lipid
 profile; hemoglobin A1c levels (HbA1C); free fatty acids, insulin and C-peptide
 concentrations and for a pregnancy test (females only). An EKG also will be performed. All
 diabetic patients who are deemed healthy and meet eligibility criteria (see below) based upon
 physical exam, medical history and screening laboratory tests will be invited to participate
 in the study.

 Subjects must meet all of the following criteria: 1) male or female 18-65 years of age; 2)
 type 2 diabetes based on the American Diabetes Association criteria; 3) HbA1c = 6.5-9.0%
 while on diet alone or diet plus sulfonylurea (or meglitinides) therapy; 4) no history of
 thiazolidinediones, insulin, ACE inhibitor or AII-receptor blockade therapy; 5) taking no
 medications known to affect glycemic control or endothelial function, unless the medication
 has been stable for at least 3 months; 6) blood pressure equal or below 140/90 mmHg; 7) not
 pregnant and willing to take appropriate contraceptive measures if capable of becoming
 pregnant; 8) serum creatinine below 1.7 mg/dl in female and 1.8 mg/dl in males; 9) ALT or AST
 less than 2 times the upper limit of normal for the laboratory and absence of clinical signs
 or symptoms of liver disease; 10) hematocrit > 34% in females and >35% in males; 11) normal
 thyroid function; 12) no evidence of coronary heart disease (by history or EKG) or moderate
 to severe congestive heart failure (NY Heart Association Cardiac Class III or IV); 13) no
 history or the presence of any clinically significant or unstable medical condition that
 makes the subject unlikely to complete the study in the opinion of the PI; and 14) absence of
 any condition or situations which would preclude adherence and completion of the protocol;
 15) the ability to give voluntary informed consent. All clinical laboratory analyses for this
 trial will be performed at the central laboratory at the University of Texas Health Science
 Center in San Antonio. All research analytical determinations will be performed in the
 laboratories of the Diabetes Division. Eligible subjects will return one week after Visit 1
 for a second visit (Visit 2), a subject number will be assigned, and a nutritional assessment
 by a dietitian will be performed. A morning urine specimen will be collected for the
 determination of microalbumin-to-creatinine ratio. Patients also will have baseline
 determinations of circulating plasminogen activator inhibitor PAI-1, endothelin-1, VCAM,
 ECAM, p-selectin and e-selectin (markers of endothelial damage), C-reactive protein and
 interleukins (markers of inflammation); PAI-1 (marker o hypercoagulable state); plasma lipids
 (total HDL/LDL cholesterol and triglycerides), as well as LDL and HDL particle size, and
 apolipoproteins A1, B and C-III. All personal data, anthropometric measurements (height,
 weight, waist-to-hip ratio), dietary history, vital signs and clinical findings will be
 recorded in a confidential master file.

 Euglycemic Insulin Clamp with Muscle Biopsies. The degree of insulin resistance will be
 estimated with the euglycemic hyperinsulinemic clamp technique (23) using an insulin infusion
 rate (80 mU/m2.min), as previously described (24). Briefly, one week after completion of the
 screening visit 2 (Visit 3), patients will report to the Clinical Research Unit at 8:00 AM
 after a 10-12 hour overnight fast. Catheters will be inserted into a peripheral vein (for
 infusion of all test substances) and into a retrograde hand vein (for blood sampling), which
 will be placed in a heated box (750 C). At time t = -120 minutes, a percutaneous biopsy of
 the vastus lateralis muscle will be performed under local anesthesia as previously described
 (25). The subject then will be allowed to rest in bed for 60 minutes. At time t = -30 min to
 0 min baseline blood samples will be obtained. At time = 0 min, insulin will be infused at 80
 mU/m2.min for 180 minutes to raise the plasma insulin concentration to approximately 120
 µU/ml to examine peripheral tissue (muscle) sensitivity to insulin. During the insulin clamp,
 a small amount of blood (0.5 ml) is withdrawn every 5 minutes from the arterialized venous
 blood (heated box technique) to determine plasma glucose concentration. When the plasma
 glucose concentration declines to 100 mg/dl a variable infusion of 20% dextrose is
 appropriately adjusted to maintain the plasma glucose concentration constant at this level
 (±5%). The rate of exogenous glucose infusion will be averaged during the last 60 minutes to
 provide a quantitative measure of insulin-mediated (primarily muscle) total glucose disposal.
 Insulin infusion will be stopped and the study will be terminated at t = 180 minutes.
 Patients will be observed for an additional 90 minutes after consuming a meal and they will
 be allowed to leave after plasma glucose is stable.

 Vascular Studies. Subjects will be asked to return to the Clinical Research Unit one week
 after the euglycemic insulin clamp for vascular studies (Visit 4). Change in forearm blood
 flow following brachial artery infusion of acetylcholine (endothelial NO-dependent) and
 sodium nitroprusside (endothelial-independent) will be used to assess the arterial
 vasodilatory response to physiological stimuli. All test substances will be infused into the
 non-dominant arm. After local skin anesthesia with lidocaine, a 20-gauge needle attached to
 an arterial line (Arrow International, Erding, Germany) will be used to cannulate the
 brachial artery. A pressurized 0.9% saline solution bag will be connected to the arterial
 line, and a constant drip will be started at t = -60 min to keep the arterial line patent
 throughout the study. Subjects will then be allowed to rest for 60 minutes. At time t = -5
 min, five consecutive measurements of forearm blood flow will be performed using a mercury
 strain gauge plethysmography (Hokanson Plethysmograph, Model EC4, Hokanson, Bellevue, WA),
 combined with a rapid cuff inflator. Test substances will be infused according to a variation
 of the original design of Bergholm et al, as follows. After completion of baseline
 measurements, an intra-brachial infusion of sodium nitroprusside at 3 microg/min will be
 started and continued for 12 minutes (the last 6 minutes are used to establish the local
 vascular effect of sodium nitroprusside). At time t = 12 min, the infusion rate of sodium
 nitroprusside is increased to 10 microg/min and the infusion continued for an additional 6
 minutes. At time t = 18 min, the sodium nitroprusside infusion is stopped and 2 ml of blood
 is withdrawn to clear the arterial line. Saline is then infused for 30 minutes and at time t
 = 48 min, an intra-brachial infusion of acetylcholine at a constant rate of 15 microg/min is
 started and continued for 8 minutes (the last 6 minutes are used to establish the local
 vascular effect of acetylcholine). At time t = 54 min, the infusion rate of acetylcholine is
 doubled to 30 microg/min and the infusion continued for and additional 6 minutes. Forearm
 blood flow is measured five consecutive times during the last 3 minutes of each
 intra-brachial infusion period, i.e., at time t = 9-12 min and t = 15-18 min, respectively
 during the low and high nitroprusside dose, and at time t = 51-54 min and t = 57-60 min,
 respectively during the low and high acetylcholine dose. At time t= 60 min, the acetylcholine
 infusion is stopped and the study terminated. At the end of the experiment the arterial line
 is removed and compression to the area applied for 10 minutes. The radial pulse is verified
 every 5 minutes during the 30-min period after the experiment is terminated, before subjects
 are allowed to leave. Subjects will be contacted within 24 hrs and 1week after completion of
 the study to ascertain that there have been no complications.

 Upon completion of baseline metabolic and vascular studies patients will be started on
 pioglitazone, 30 mg per day (Visit 6). Patients will be asked to continue with their previous
 medical regimen and will be followed by the PI/nursing team at the Texas Diabetes Institute.
 All participants will be contacted weekly by phone to ascertain that there are no adverse
 events during the initiation period of 4 weeks, at which time the dose of pioglitazone will
 be increased to 45 mg per day. All patients will be required to return for office visits at 6
 weeks intervals during the entire 6 months of the study (Visits 7-10). All available records
 of capillary blood glucose, adverse events and other major events will be collected and added
 to a confidential file under the patients' assigned number. Office visits every six weeks are
 intended to review patients' interim medical history to document adverse events, to emphasize
 adherence to the medical regimen, to obtain blood for laboratory determinations, and record
 vital signs, body weight, neurologic and fundoscopic findings. Blood and urine measurements
 similar to those in Visit 2 also will be obtained. Metabolic and vascular studies similar to
 those described above (Visits 3,4,5) before the initiation of pioglitazone therapy will be
 performed again at 3 and 6 months (Visits 8 and 10) and insulin clamps with muscle biopsies
 will be repeated at the end of the 6 month period (Visit 10). Patients will be discontinued
 from the study program for any of the following reasons: 1) the principal investigator
 decides that the subject should be withdrawn, either because of serious adverse events or
 clinically significant laboratory abnormalities; 2) the subject requests that he/she be
 withdrawn; 3) the subject becomes pregnant during the study; and 4) the subject is
 non-compliant with study guidelines, recommendations, and for the treatment regimen. Early
 termination at any time will be accompanied by collection of data as outlined in Visit 10.
 Any patient who is withdrawn from the study will be followed for as long as necessary to
 establish a stable therapeutic program.

 Laboratory Tests. Measurements of fasting plasma glucose (Beckman Glucose Analyzer), HbA1c
 (32) insulin (33) and C-peptide (34) by radioimmunoassay, and free fatty acids by HPLC (35)
 will be performed in the principal investigator's laboratory at the Texas Diabetes Institute,
 as previously described. Assays for VCAM and ICAM (3), p-Selectin and s-Selectin (4), PAI-1
 and endothelin-1 (36), and plasma lipids by MRS (37) and apolipoproteins (38) will be
 performed in the laboratories of the Diabetes Division at the University of Texas Health
 Science Center. Measurements of IRS-1 thyrosine phosphorylation, PI-3 kinase activity and
 association of PI-3 kinase with IRS-1 also will be performed in the Diabetes Division
 laboratory.

 Sample Size and Data Analysis Methods. We will perform correlation analyses to examine the
 relationships between the increment in skin and forearm blood flow following acetylcholine
 infusion and the improvements in plasma hemoglobin A1c levels and whole body insulin
 sensitivity before and after pioglitazone therapy using ANOVA with repeated measures over
 time. We anticipate that pioglitazone treatment will improve glycemic control and tissue
 insulin sensitivity and that the improvements in hemoglobin A1c and insulin action will be
 closely related to improvements in acetylcholine-stimulated increments in skin and forearm
 blood flow. Changes in insulin sensitivity (insulin clamp data), insulin signaling pathways
 activity, nitric oxide production in vivo, endothelial function, FPG and HbA1c will be
 compared. Further, we anticipate that plasma levels of endothelial damage markers
 (ICAM/VCAM), p-Selectin, s-Selectin and PAI-1 and inflammatory markers (C-reactive protein
 and interleukins), which are elevated in diabetic patients, will be reduced following
 pioglitazone, and that the reduction will correlate with the improvements in skin and muscle
 endothelial function. We also expect to observe an improvement in the plasma lipid profile,
 especially triglycerides and free fatty acids after pioglitazone treatment. Similar
 correlations between improved endothelial-independent blood flow (nitroprusside-stimulated)
 following pioglitazone treatment and enhanced insulin sensitivity, glycemic control, nitric
 oxide production and insulin signaling pathways will also be performed. We expect that
 improved insulin sensitivity (insulin clamp) and increased NO production in vivo following
 pioglitazone treatment will be closely related to enhanced IRS-1 tyrosine phosphorylation and
 total IRS-1 associated PI-3 kinase activity, which we previously have shown to be markedly
 impaired in type 2 diabetic individuals (39). In contrast, we expect that the MAP-kinase
 (mitogenic) pathway, which regulates vascular smooth muscle cell growth/proliferation and
 which we have shown to be normal or upregulated in type 2 diabetics, may be inhibited
 following pioglitazone treatment. It would be of great pathogenic significance if reduction
 in the MAP kinase activity correlated with the improved endothelial function following
 pioglitazone therapy.

 The sample size calculation was based upon the changes in forearm blood flow in response to
 acetylcholine and sodium nitroprusside in normal and diabetic subjects during preliminary
 studies and from previously published studies by Caballero et al (10). The pooled standard
 deviation for changes from baseline to endpoint in forearm blood flow (ml/100gr per min) in
 our laboratory is 1.20. In order to detect with 90% power a difference in forearm blood flow
 in response to these physiologic agents before and after 6 months of pioglitazone therapy, 40
 completed patients are required. Assuming a dropout rate of approximately 20%, 50 diabetic
 patients must be recruited to obtain 40 completers. Based on the laser Doppler skin blood
 flow measurements, we have an 80% chance of showing an improvement of ~50% in skin blood in
 response to physiologic stimuli (from 65% to 100%) following pioglitazone therapy. Thus, we
 will have more than sufficient diabetic individuals to detect statistically significant
 changes in skin and muscle blood flow.
Inclusion Criteria:

 1. male or female 18-65 years of age;

 2. type 2 diabetes based on the American Diabetes Association criteria;

 3. HbA1c = 6.5-9.0% while on diet alone or diet plus sulfonylurea (or meglitinides)
 therapy;

 4. no history of thiazolidinediones, insulin, ACE inhibitor or AII-receptor blockade
 therapy;

 5. taking no medications known to affect glycemic control or endothelial function, unless
 the medication has been stable for at least 3 months;

 6. blood pressure equal or below 140/90 mmHg;

 7. not pregnant and willing to take appropriate contraceptive measures if capable of
 becoming pregnant;

 8. serum creatinine below 1.7 mg/dl in female and 1.8 mg/dl in males;

 9. ALT (SGTP) or AST (SGOT) less than 2 times the upper limit of normal for the
 laboratory and absence of clinical signs or symptoms of liver disease;

 10. hematocrit > 34% in females and >35% in males;

 11. normal thyroid function;

 12. no evidence of coronary heart disease (by history or EKG) or moderate to severe
 congestive heart failure (NY Heart Association Cardiac Class III or IV);

 13. no history or the presence of any clinically significant or unstable medical condition
 that makes the subject unlikely to complete the study in the opinion of the PI; and

 14. absence of any condition or situations which would preclude adherence and completion
 of the protocol;

 15. the ability to give voluntary informed consent.

 Exclusion Criteria:

 1. Subjects were excluded from study if they had ever received insulin, metformin, TZDs,
 exenatide or DPP IV inhibitor.

 2. All subjects were free of cardiovascular, renal or major organ disease, as determined
 by medical history, physical examination, screening blood chemistries, complete blood
 cell count, and electrocardiogram.