Top PDF Novel gut-based pharmacology of metformin in patients with type 2 diabetes mellitus.

Novel gut-based pharmacology of metformin in patients with type 2 diabetes mellitus.

Novel gut-based pharmacology of metformin in patients with type 2 diabetes mellitus.

Metformin, a biguanide derivate, has pleiotropic effects beyond glucose reduction, including improvement of lipid profiles and lowering microvascular and macrovascular complications associated with type 2 diabetes mellitus (T2DM). These effects have been ascribed to adenosine monophosphate-activated protein kinase (AMPK) activation in the liver and skeletal muscle. However, metformin effects are not attenuated when AMPK is knocked out and intravenous metformin is less effective than oral medication, raising the possibility of important gut pharmacology. We hypothesized that the pharmacology of metformin includes alteration of bile acid recirculation and gut microbiota resulting in enhanced enteroendocrine hormone secretion. In this study we evaluated T2DM subjects on and off metformin monotherapy to characterize the gut-based mechanisms of metformin. Subjects were studied at 4 time points: (i) at baseline on metformin, (ii) 7 days after stopping metformin, (iii) when fasting blood glucose (FBG) had risen by 25% after stopping metformin, and (iv) when FBG returned to baseline levels after restarting the metformin. At these timepoints we profiled glucose, insulin, gut hormones (glucagon-like peptide-1 (GLP-1), peptide tyrosine-tyrosine (PYY) and glucose-dependent insulinotropic peptide (GIP) and bile acids in blood, as well as duodenal and faecal bile acids and gut microbiota. We found that metformin withdrawal was associated with a reduction of active and total GLP-1 and elevation of serum bile acids, especially cholic acid and its conjugates. These effects reversed when metformin was restarted. Effects on circulating PYY were more modest, while GIP changes were negligible. Microbiota abundance of the phylum Firmicutes was positively correlated with changes in cholic acid and conjugates, while Bacteroidetes abundance was negatively correlated. Firmicutes and Bacteroidetes representation were also correlated with levels of serum PYY. Our study suggests that metformin has complex effects due to gut-based pharmacology which might provide insights into novel therapeutic approaches to treat T2DM and associated metabolic diseases.
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Effect of oral hypoglycaemic agents on bone metabolism in patients with type 2 diabetes mellitus

Effect of oral hypoglycaemic agents on bone metabolism in patients with type 2 diabetes mellitus

Diabetes mellitus (DM) and osteoporosis are the two important public health problems in India. The burden of both these conditions is expected to increase in the near future in view of changing lifestyle habits and ageing population. Indians are at risk of osteoporosis due to their low body mass index (BMI), genetic predisposition and nutritional factors. The diseases type 1 DM and type 2 DM (T2DM) are associated with increased fracture risk in the disease population, in spite of difference in the bone mineral density (BMD). An increase in fracture risk is also reported among older patients with T2DM despite frequently reported normal or increased BMD. Administration of insulin stimulates osteoblast activity and bone mineral apposition rates. The impact of endogenous insulin production, insulin sensitivity, and exogenous insulin administration as an anabolic agent for bone in T2DM has not been clarified. Biguanides and sulphonylureas do not appear to have adverse effects on BMD. Preclinical evidence suggests that incretin-based drugs may be beneficial for bone, but clinical evidence to support this hypothesis is not yet available. Thiazolidinedione (TZD) group of agents have been implicated in causing osteoporosis in various animal studies and some human studies available till date. The debate regarding this is issue is still ongoing. Randomized controlled studies with larger sample size preferably involving multiple centres, multiple ethnicities are required to answer these queries.
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Rev. Assoc. Med. Bras.  vol.58 número3 en v58n3a08

Rev. Assoc. Med. Bras. vol.58 número3 en v58n3a08

Objectives: To compare costs and clinical beneits of three additional therapies to met- formin (MF) for patients with diabetes mellitus type 2 (DM2). Methods: A discrete event simulation model was built to estimate the cost-utility ratio (cost per quality-adjusted life years [QALY]) of saxagliptine as an additional therapy to MF when compared to rosiglitazone or pioglitazone. A budget impact model (BIM) was built to simulate the economic impact of saxagliptine use in the context of the Brazilian private health sys- tem. Results: he acquiring medication costs for the hypothetical patient group analyzed in a time frame of three years were R$  10,850,185, R$  14,836,265 and R$ 14,679,099 for saxagliptine, pioglitazone and rosiglitazone, respectively. Saxagliptine showed lower costs and greater efectiveness in both comparisons, with projected savings for the irst three years of R$ 3,874 and R$ 3,996, respectively. he BIM estimated cumulative savings of R$ 417,958 with the repayment of saxagliptine in three years from the perspective of a health plan with 1,000,000 covered individuals. Conclusion: From the perspective of private paying source, the projection is that adding saxagliptine with MF save costs when compared with the addition of rosiglitazone or pioglitazone in patients with DM2 that have not reached the HbA1c goal with metformin monotherapy. he BIM of including saxagliptine in the reimbursement lists of health plans indicated signiicant savings on the three-year horizon.
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Dapagliflozin versus saxagliptin as add-on therapy in patients with type 2 diabetes inadequately controlled with metformin

Dapagliflozin versus saxagliptin as add-on therapy in patients with type 2 diabetes inadequately controlled with metformin

cotransporter-2 (SGLT2) inhibitor, 5) a glucagon-like peptide-1 (GLP-1) receptor agonist, or 6) a basal insulin analogue, as an add-on therapy when the individualized HbA 1c target is not achieved after ∼3 months of treatment with metformin alone (1). The ADA/EASD position statement does not recommend any specific preference for a drug to be used as a dual therapy and instead suggests that the drug choice should be individualized based on patient preferences, hypoglycemia risk, side effect profile, and cost, in addition to other patient and disease characteristics (2). The ADA/EASD position statement further details that other drugs such as α-glucosidase inhibitors, colesevelam, bromocriptine and pramlintide may be used in specific situations but are generally not preferred due to their modest efficacy, side-effect profiles and dosing frequency.
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Dose- and time-dependent association of smoking and its cessation with glycemic control and insulin resistance in male patients with type 2 diabetes mellitus: the Fukuoka Diabetes Registry.

Dose- and time-dependent association of smoking and its cessation with glycemic control and insulin resistance in male patients with type 2 diabetes mellitus: the Fukuoka Diabetes Registry.

pancreatic fibrosis [28]. On the basis of these reports, the adverse effects of cigarette smoking on glycemia seem to be mediated through both insulin resistance and impaired insulin secre- tion. In this study, HOMA2-IR levels showed a dose-response relation with the increasing amount of smoking (Table 5). Estimates of insulin resistance derived from HOMA is an indi- rect parameter compared with glucose clamp tests, a golden standard. However, it may be ap- propriate for use in large epidemiological studies [29], attributable to its simple and convenient nature. This use in diabetic patients showed a strong correlation with the insulin resistance index assessed by euglycemic–hyperinsulinemic clamp (r = −0.725, P <0.0001) [30]. Another study also showed a strong correlation between clamp-measured total glucose disposal and HOMA-estimated insulin sensitivity (r = −0.820, P <0.0001) in subjects including diabetic pa- tients, with no substantial difference between nondiabetic (r = −0.754) and diabetic patients (r = −0.695) [31]. Though the cautious interpretation is needed, the use of HOMA model in subjects treated by insulin or insulin secretagogues may be applicable [29]. Some prior studies reported the relationship between smoking and insulin resistance, but the results were incon- sistent [10,11,18,26]. In an experimental study among 40 healthy volunteers [18], smokers pre- sented higher plasma insulin concentrations in response to oral glucose load despite similar glucose concentrations, and higher steady-state plasma glucose concentration compared with never smokers. The smoking-insulin resistance association was also observed in diabetic pa- tients in earlier studies, though the numbers of subjects were relatively small (40 [10] and 52 patients [11], respectively). In contrast, another community-based cross-sectional study in Sweden showed no association between smoking status and insulin resistance [26]. The dose- response relationship of our study
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Relationship of glycated hemoglobin levels with myocardial injury following elective percutaneous coronary intervention in patients with type 2 diabetes mellitus.

Relationship of glycated hemoglobin levels with myocardial injury following elective percutaneous coronary intervention in patients with type 2 diabetes mellitus.

more and more diabetic patients receive PCI. However, PCI was still frequently companied with postprocedural cardiac marker elevation. There was a large body of data correlating troponin elevation after elective PCI with adverse clinical outcomes [12– 15]. Third universal definition of myocardial infarction has raised the diagnostic threshold of PCI-related myocardial infarction from the elevation of troponin above 3 times ULN to the elevation of troponin above 5 times ULN, and suggested that this threshold was arbitrarily chosen, based on clinical judgement and societal implications of the label of PCI-related myocardial infarction. Myocardial injury is used for postprocedural cTn value is . 16ULN and #56ULN [16]. So, we used many different cTnI cut points. Although a number of studies have investigated the risk factors associated with periprocedural myocardial infarction or injury [9,17,18], less of them focused on diabetic patients or the impact of glycemic control on periprocedural myocardial infarc- tion or injury in diabetic patients. And elevated HbA1c or poor glycemic control is associated with increased risk of cardiovascular events in diabetic patients [4–6], but whether elevated HbA1c is still associated with increased risk of myocardial infarction or injury following elective PCI in diabetic patients is still unknown. In the present study, we included 994 diabetic patients undergoing elective PCI to determine the relation of preprocedural HbA1c levels with postprocedural cTnI elevation. Univariate analysis showed that some clinical and procedural characteristics
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Association of glycemic control with progression of diabetic retinopathy in type 2 diabetes mellitus patients in Malaysia

Association of glycemic control with progression of diabetic retinopathy in type 2 diabetes mellitus patients in Malaysia

The mean fasting blood glucose was lower in the PDR compared to others stages of diabetic retinopathy and DME. This result contradicts a previous study by Teuscher et al which showed that the incidence of PDR was higher in patients with FBG of more than 14.5mmol/L, as well as NPDR (Teuscher, Schnell, Wilson, 1988). Besides that, a study conducted in United Kingdom reported that, mean fasting blood glucose, 8.51mmol/L with intensive therapy that decreased the mean of FBG around 1.2mmol/L was associated with reduction in the progression of DR (Patel et al., 2008). Fasting blood glucose is different from glycated haemoglobin concentration as it can be varied, affected by diet taken and fasting state before the test done. Therefore, the discrepancy might be because there was variation in the way of data collection. Moreover, the data for study conducted by Teuscher, Schnell and Wilson (1998) and Patel et al. (2008) was based on the baseline
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RETINOPATHY IN TYPE 2 DIABETES MELLITUS AND SERUM MAGNESIUM LEVELS

RETINOPATHY IN TYPE 2 DIABETES MELLITUS AND SERUM MAGNESIUM LEVELS

J of Evidence Based Med & Hlthcare, pISSN- 2349-2562, eISSN- 2349-2570/ Vol. 2/Issue 6/Feb 09, 2015 Page 682 increasing magnesium levels with age were probably due to impaired renal function and the sample size, (87 diabetics, 30 non-diabetics) was relatively small to confirm male preponderance. In our study, patients with impaired renal functions were excluded. Our results confirm to the recent reports that have not shown any significant associations between sex, age and duration of diabetes with serum magnesium levels. 18,19
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Vascular complications of black patients with type 2 diabetes mellitus in Southern Brazil

Vascular complications of black patients with type 2 diabetes mellitus in Southern Brazil

A total of 1810 patients with DM2 were analyzed from a multicenter study that began recruiting patients in Brazil in 2002. The study was designed to identify factors involved in the development of chronic micro- and macrovascular complications of DM, and included 4 centers at general hospitals in the State of Rio Grande do Sul, Brazil. All patients with DM2 attending the Endocrine Clinics and dialysis units of these hospitals were invited to participate. Inclusion criteria were: DM2 according to World Health Organization (WHO) criteria (age ≥35 years and absence of insulin requirement in the first 5 years after diagnosis). Chronic diabetic complications (DR, DN, and distal sen- sory neuropathy, DSN) were assessed in the entire sam- ple. Information about macrovascular disease was avail- able for 1177 patients. Ethnicity was classified based on self-reported skin color and recorded as white, black, white-black admixture (mixed) and “other”. Mixed subjects (N = 181) were excluded from this analysis because of the limitation of this definition (24). Subjects with “other” ethnic
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Arq Bras Endocrinol Metab  vol.55 número4

Arq Bras Endocrinol Metab vol.55 número4

Objective: To evaluate the effectiveness of adding vildagliptin to the treatment of patients with inadequately controlled type 2 diabetes mellitus (T2DM) treated with a combination of metformin and a sulphonylurea. Subjects and methods: 37 T2DM patients with HbA1c ranging from 7.7% to 12.4% (mean of 9.30 ± 1.38), despite the use of metformin in combination with a sulphonylurea, were additionally treated with vildagliptin (100 mg/day) for at least 6 months. Results: During triple oral therapy (TOT) HbA1c levels < 7% were achieved in 11 patients (29.7%), whereas levels of fasting plasma glucose (FPG) < 120 mg/dL were observed in 12 patients (32.4%). Both findings were observed in 10 patients (27.0%). Compared to nonresponsive subjects, lower mean baseline HbA1c and FPG levels were seen in responsive patients, but the difference was only statistically significant for fasting plasma glucose (FPG). Moreover, there was considerable overlap between the two groups. Conclusion: Our preliminary results suggest that TOT with metformin, a sul- phonylurea and vildagliptin may be useful for some T2DM patients nonresponsive to combina- tion therapy with metformin and sulphonylurea. Arq Bras Endocrinol Metab. 2011;55(4):260-5
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Medication adherence in type 2 diabetes mellitus patients: a cross-sectional study

Medication adherence in type 2 diabetes mellitus patients: a cross-sectional study

Several limitations of this study need to be outpoin- ted. All our patients were recruited from one primary care setting, and thus might not be representative of the general diabetic population in Portugal. The use of self- report data on medication adherence may have a ten- dency to overestimate adherence, due to recall biases and social desirability. Moreover, the exclusion of anal- phabet patients who were not accompanied by someone of their trust to help them answering the questionnaire may be an important bias. On the other hand, our sam- ple is based on the patients present in a primary care ap- pointment with their doctor. We notice a deviation in ge- neral characteristics from those we could expect from the diabetic population of an entire health center, but it does not seem to interfere in the conclusion about the
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Long-Term Effectiveness and Cost-Effectiveness of Metformin Combined with Liraglutide or Exenatide for Type 2 Diabetes Mellitus Based on the CORE Diabetes Model Study.

Long-Term Effectiveness and Cost-Effectiveness of Metformin Combined with Liraglutide or Exenatide for Type 2 Diabetes Mellitus Based on the CORE Diabetes Model Study.

This study is not without limitations. 1) The sample size was relatively small and some of the measurements for the CORE model could be collected and supplemented by other sources, which might affect the accuracy of our findings. In addition, many studies used the CORE model for Asian populations [8,25–28], but the model has never been validated for Asian pop- ulation. Additional studies with a larger sample size and longer follow-up are still necessary to confirm these findings. 2) The none-measurable costs such as depreciation charge for medical equipment, indirect medical costs, disease onset and death costs, and other intangible costs including pain and sadness were not included into the study. 3) The present study is based on epidemiological studies (UKPDS, Fei Minghan Heart Study, and DCCT), which may result in bias. Therefore, prospective data are needed to improve the epidemiological data for the valida- tion of the effectiveness of the CORE model in China. 4) Drug cost and medical equipment depreciation costs also need to be analyzed in future studies. 5) Finally, further studies are needed to confirm the BMI results.
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EVALUATION OF TESTOSTERONE DEFICIENCY IN PATIENTS OF TYPE 2 DIABETES MELLITUS

EVALUATION OF TESTOSTERONE DEFICIENCY IN PATIENTS OF TYPE 2 DIABETES MELLITUS

The mechanisms underlying hypogonadotropic hypogonadism in men with type 2 diabetes are not clear. It has been suggested that an excessive increase in fat mass may result in an increase in the activity of aromatase enzyme, which causes greater conversion of testosterone into oestrogen (The primary female sex hormone). An increase in oestrogen levels would lead to the suppression of gonadotropin releasing hormone and impaired secretion of gonadotropin by the pituitary gland. This results in the reduction of both testosterone secretion and mature sperm production. The state of hypogonadotropic hypogonadism is not entirely dependent upon obesity. 6 Based on animal studies
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Gut hormone pharmacology of a novel GPR119 agonist (GSK1292263), metformin, and sitagliptin in type 2 diabetes mellitus: results from two randomized studies.

Gut hormone pharmacology of a novel GPR119 agonist (GSK1292263), metformin, and sitagliptin in type 2 diabetes mellitus: results from two randomized studies.

GPR119 receptor agonists improve glucose metabolism and alter gut hormone profiles in animal models and healthy subjects. We therefore investigated the pharmacology of GSK1292263 (GSK263), a selective GPR119 agonist, in two randomized, placebo-controlled studies that enrolled subjects with type 2 diabetes. Study 1 had drug-naive subjects or subjects who had stopped their diabetic medications, and Study 2 had subjects taking metformin. GSK263 was administered as single (25–800 mg; n = 45) or multiple doses (100–600 mg/day for 14 days; n = 96). Placebo and sitagliptin 100 mg/day were administered as comparators. In Study 1, sitagliptin was co-administered with GSK263 or placebo on Day 14 of dosing. Oral glucose and meal challenges were used to assess the effects on plasma glucose, insulin, C-peptide, glucagon, peptide tyrosine-tyrosine (PYY), glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). After 13 days of dosing, GSK263 significantly increased plasma total PYY levels by ,five-fold compared with placebo, reaching peak concentrations of ,50 pM after each of the three standardized meals with the 300 mg BID dose. Co-dosing of GSK263 and metformin augmented peak concentrations to ,100 pM at lunchtime. GSK263 had no effect on active or total GLP-1 or GIP, but co-dosing with metformin increased post-prandial total GLP-1, with little effect on active GLP-1. Sitagliptin increased active GLP-1, but caused a profound suppression of total PYY, GLP-1, and GIP when dosed alone or with GSK263. This suppression of peptides was reduced when sitagliptin was co-dosed with metformin. GSK263 had no significant effect on circulating glucose, insulin, C-peptide or glucagon levels. We conclude that GSK263 did not improve glucose control in type 2 diabetics, but it had profound effects on circulating PYY. The gut hormone effects of this GPR119 agonist were modulated when co-dosed with metformin and sitagliptin. Metformin may modulate negative feedback loops controlling the secretion of enteroendocrine peptides.
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Diabetes Mellitus: terapêutica e estratégias de prevenção

Diabetes Mellitus: terapêutica e estratégias de prevenção

A   resistência   à   insulina   é   o   sinal   metabólico   anormal   mais   precoce   antes   do   desenvolvimento   da   DMT2,   (26)   que   poderá   estar   presente   cerca   de   uma   década   antes  do  aparecimento  da  hiperglicemia  pós-­prandial  e/ou  em  jejum.  A  manutenção   dos   níveis   normais   de   glicose   durante   esse   tempo   explica-­se   pelo   aumento   da   produção   de   insulina   resultante   de   uma   maior   atividade   das   células-­beta   pancreáticas  que  compensa  a  resistência  dos  tecidos  à  insulina  (27).  Esta  atividade   compensatória  por  parte  das  células-­beta  do  pâncreas  leva  à  sua  perda  progressiva   e,   portanto,   como   resultado   dá-­se   um   aumento   progressivo   da   concentração   de   glicose   no   sangue,   inicialmente   manifestada   como   hiperglicemia   intermédia.   À   medida   que   a   doença   evolui   mais   células   se   perdem,   maior   a   glicémia   e   eventualmente  ocorre  a  diabetes  (24).  A  exposição  crónica  a  altas  concentrações  de   glicose   origina   espécies   reativas   de   oxigénio   causando   stress   oxidativo,   o   que   aumenta  a  produção  de  ácidos  gordos  livres.  Essas  substâncias  contribuem  para  a   deterioração  da  função  das  células-­beta  e  para  a  sua  apoptose  (24).    
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CORRELATION OF GLYCOSYLATED HEMOGLOBIN AND BMI IN TYPE 2 DIABETES MELLITUS  PATIENTS

CORRELATION OF GLYCOSYLATED HEMOGLOBIN AND BMI IN TYPE 2 DIABETES MELLITUS PATIENTS

They represent the current glycemic status of the patient and they are poor indicators of long-term control of diabetes. For the last few years, estimation of glycosylated hemoglobin and fructosamine has been increasingly used to achieve better monitoring of long-term glycemic control in diabetics.

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Table 3 - Meta-analysis on the relative risk (RR) of cancer in

Table 3 - Meta-analysis on the relative risk (RR) of cancer in

side effects of high doses (99,110,111). More than 40 phase I/II clinical cancer trials (http://clinicaltrials.gov/) on metformin in combination with chemotherapeutics are under- way worldwide. These studies are examining the antineo- plastic effects of metformin. This drug is used jointly with chemotherapeutic agents in cancers of the digestive (hepatic, gastric, pancreatic, and colorectal) and reproductive systems (ovarian and endometrial) and lung, prostate, and breast cancer, and the clinical limitations of metformin in cancer treat- ment are being determined. Several trials have reported synergistic or additive effects of such combinations (87,111-131). In contrast, observational retrospective studies have observed that these combinations are antagonistic in certain cancers (132-136). Therefore, metformin synergizes with standard chemotherapy drugs to increase chemosensitivity in specific cancers.
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CHARACTERIZATION OF TYPE 2 MELLITUS DIABETES PATIENTS ASSISTED IN AN ENDOCRINOLOGY CLINIC

CHARACTERIZATION OF TYPE 2 MELLITUS DIABETES PATIENTS ASSISTED IN AN ENDOCRINOLOGY CLINIC

This study aims to study the characterization of type 2 diabetic patients from an endocrinology clinic. This is a descriptive study of a quantitative approach, performed with 252 elderly people assisted in the endocrinology ward of the University Hospital of the Federal University of Maranhão. The results showed that the patients were mostly female (67.46%), elderly (63.50%), brown (57.94%), retired (57.93%), (38.89%), with a monthly income of 1 to 2 minimum wages (67.46%), lived in other municipalities of the island (São José de Ribamar, Raposa and Paço do Lumiar) (49.21%). Smokers (61.90%), did not use alcohol (56.35%), and had comorbidity arterial hypertension (56.35%). The objectives of the present study were reached, allowing to conclude that the profile of patients in this population is similar to previous studies. Thus, it can be highlighted that the majority of patients assisted at the clinic is female, elderly, retired and of brown color, with income between 1 and 2 minimum wages, most of whom finished high school.
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Comparison of Vildagliptin-Metformin and Glimepiride-Metformin Treatments in Type 2 Diabetic Patients

Comparison of Vildagliptin-Metformin and Glimepiride-Metformin Treatments in Type 2 Diabetic Patients

he present study was randomized, open-label and compara- tive. Patients who attended the outpatient diabetic clinic of Chungbuk National University Hospital were included, and exclusion criteria were evaluated during screening (week -2, visit 1). Type 2 diabetic patients with HbA1c levels greater than 7.0% who were naïve or were receiving monotherapy with oral hypoglycemic agents such as glimepiride (2 to 4 mg) or met- formin (500 to 1,000 mg) for less than six months prior to the visit were eligible to participate in the present study. All the patients who received previous medications had to undergo a wash-out period of at least two weeks. Patients with a history of diabetic ketoacidosis, clinically signiicant liver or renal dis- ease, congestive heart failure requiring pharmacological treat- ment, coronary artery percutaneous intervention or unstable angina within the past six months, and those over 80 years of age were excluded from the present study. Any of the follow-
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Arq Bras Endocrinol Metab  vol.50 número5

Arq Bras Endocrinol Metab vol.50 número5

All patients (242 men, 402 women; mean age at examination 58.3 ± 11.2 years; mean age at onset 47.0 ± 11.9 years) underwent standardized clinical and laboratory investigations. Weight and height were used to calculate BMI. Sitting BP was measured after a 5-min rest using a mercury sphygmomanometer. The mean value of the two measurements was used to calculate systolic and diastolic BP. Hypertension was defined as BP levels ≥ 140/90 mmHg or the use of anti-hypertensive drugs. Diabetic retinopathy was assessed by an ophthalmologist and classified as absent, non-proliferative, or proliferative. The renal status was based on the albumin excretion rate (AER) measured in at least two out of three consecutive 24-h timed urine collections. Patients were classified as normo- (AER< 20 mg/min), micro- (AER 20–199 mg/min), or macroalbuminurics (AER > 200 mg/min). Patients also answered a questionnaire about familial history of type 2 DM (parent, aunts and uncles, siblings and offspring). Responses to the ques- tionnaire were requested as ‘yes’, ‘no’ or ‘don’t know’. A clear maternal or paternal history was defined as presence of relatives with type 2 DM in three consec- utive generations or through two consecutive genera- tions with at least three affected relatives.
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