11402 • The Journal of Neuroscience, December 10, 2003 • 23(36):11402–11410 Imaging Reveals Synaptic Targets of a Swim-TerminatingNeuron in the Leech CNS Adam L. Taylor,1,2 Garrison W. Cottrell,1 David Kleinfeld,3 and William B. Kristan Jr21Department of Computer Science and Engineering, 2Neurobiology Section, Division of Biological Sciences, and 3Department of Physics, University ofCalifornia, San Diego, La Jolla, California 92093
Effects of caloric restriction and low glycemic index diets associated with metformin on glucose metabolism and cortisol response in overweight/obese subjects: a case series study
Casulari et al. Diabetology & Metabolic Syndrome (2015) 7:65 DIABETOLOGY &
Effects of caloric restriction and low glycemicindex diets associated with metformin on glucosemetabolism and cortisol response in overweight/obese subjects: a case series study Luiz Augusto Casulari1,5*, Donatella Dondi2, Fabio Celotti2, Fábio Vinicius Pires da Silva3,Caio Eduardo Gonçalves Reis3 and Teresa Helena Macedo da Costa4 Background: To determine whether cortisol secretion and glucocorticoid receptors in lymphocytes and monocytesare altered in patients with impaired glucose tolerance, and whether treatment with a hypocaloric diet andmetformin could interfere with these aspects.
Methods: This is an analytical, interventional, case series study. Patients with impaired glucose tolerance wereincluded. They received 500 mg of metformin twice daily and followed a low glycemic index diet for 16 weeks.
Cortisol levels were assessed at 8:00 A.M. before and after use of 0.25 mg of dexamethasone at 11:00 P.M. the daybefore.
Results: Sixteen subjects (9 men) were included. Normal basal levels of cortisol and adequate responses to the lowdose of dexamethasone were observed before and after treatment. There was no significant correlation betweenthe parameters evaluated and cortisol levels. Nevertheless, there was a strong correlation between the number ofglucocorticoid receptors, BMI (r = 0.88; p = 0.02), and insulin AUC (r = 0.94; p = 0.005) before treatment; aftertreatment, all these associations ceased to exist.
Conclusion: The cortisol secretion remained normal in the group of patients with impaired glucose tolerance.
Treatment with metformin and diet did not change this condition. However, glucocorticoid receptor number had astrong correlation with insulin, due to insulin resistance, but this characteristic was lost after treatment.
Keywords: Diabetes, Glucose intolerance, Calorie-restricted diet, Metformin, Cortisol, Receptor tolerance test (OGTT), glycemia between 141 mg/dL and Type-2 diabetes mellitus generally affects overweight or obese individuals over 40 years old. These patients have Lifestyle changes with adequate nutrition and increased insulin resistance and deficiency . However, before physical activity, with or without treatment with metfor- type-2 diabetes mellitus is established, individuals may min, can prevent or delay the onset of type-2 diabetes show the following pre-diabetic conditions: impaired fast- mellitus . Metformin, which is an insulin action ing glucose between 100 mg/dl and 125 mg/dl, and im- sensitizer, is recommended as a first-choice oral drug for paired glucose tolerance two hours after an oral glucose the treatment of type-2 diabetes due to its preventive ac-tion in populations at risk .
In order to potentially prevent the onset of type-2 dia- * Correspondence: betes in individuals with pre-diabetic symptoms, it is im- 1Unit of Endocrinology, University Hospital Brasilia, University of Brasilia, portant to study the characteristics involved in the Brasilia, Brazil5CLINEN – Clínica de Neurologia e Endocrinologia. SCN quadra 1, bloco F, pathophysiology of this condition. A still unsettled problem Ed. America Office Tower, sala 1111, Brasília, DF 70711-905, BrazilFull list of author information is available at the end of the article 2015 Casulari et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiverapplies to the data made available in this article, unless otherwise stated.
Casulari et al. Diabetology & Metabolic Syndrome (2015) 7:65 is the diabetogenic role of cortisol in obese individuals and The study protocol was approved by the Ethics Com- in patients with metabolic syndrome .
mittee in Human Research of the School of Health Sci- Several changes in the hypothalamic-pituitary-adrenal ences at the University of Brasília, Brazil (N. 035/2004).
(HPA) axis of subjects with abdominal obesity have been All volunteers were informed of the objectives of the study described: changes in pulsatile secretion of adrenocorti- and signed a written informed consent to participate.
cotropic hormone (ACTH), increased cortisol secretionafter laboratory stress tests, hyperresponsivity of the HPA axis to peptides such as the corticotropin-releasing Participants were recruited through public announce- hormone (CRH), associated or not with arginine vaso- ments. Inclusion criteria were adults aged between 18 pressin (AVP) (for references, see There is evi- and 50 years old, of both sexes, body mass index dence of peripheral and central alterations of the HPA from 25 to 35 kg/m². Smokers, pregnant or lactating axis in patients with abdominal obesity or post-menopausal women were not included, as well The use of low doses of dexamethasone has been a as individuals who had used medication and followed strategy to detect subtle changes in the negative feedback a therapeutic diet in the last two years. Subjects with of cortisol secretion in obese individuals or in those with a fasting blood glucose value higher than 126 mg/dL metabolic syndrome who are not yet diabetic. Alterations and/or higher than 200 mg/dL 120 min after the start in glucose and insulin metabolism can also be identified of the OGTT were also excluded.
–]. Nonetheless, results are conflicting. In patients The sample size was determined using the software with metabolic syndrome, a higher concentration of corti- G*Power, version 3.1.9 (Heinrich Heine University sol has been observed after treatment with dexamethasone Düsseldorf, Düsseldorf, Germany) based on the result as compared to healthy controls ]. In obese individuals, from Tam et al. considering the morning cortisol cortisol response to dexamethasone was reported as nor- levels as the main variable. A statistical power (1-β) of mal or altered [Very few data are available on 80 % and a level of significance (α) of 5 % were considered, the effect of insulin resistance on the corticosteroid recep- which resulted in a sample of 16 subjects, as the necessary number for this study.
The objectives of this study were to determine whether All subjects had cortisol inhibition < 1.8 mg/mL at cortisol secretion is altered in a group of patients with 8:00 A.M. after the ingestion of 2 mg of dexamethasone pre-diabetic conditions and whether treatment with a (Decadron, Merck Sharp & Dohme, São Paulo, Brazil) at hypocaloric diet and metformin treatment could interfere 11:00 P.M. of the previous day, thus excluding the oc- with this process. In addition, glucocorticoid receptors on currence of endogenous Cushing's syndrome mononuclear cells–lymphocytes and monocytes–were To evaluate the responsiveness of the hypothalamic- analyzed before and after treatment to determine if the pituitary-adrenal axis to low doses of dexamethasone, a receptor expression was affected in this group of pre- cortisol analysis was conducted after administration of diabetic patients.
0.25 mg of dexamethasone at 11:00 P.M. and cortisollevels were measured at 8:00 A.M.
Subjects were also submitted to an oral 75-g glucose tol- Research design and methods erance test after an overnight fast. Blood samples were Study design and ethics collected from the antecubital vein at 0, 30, 60, 90 and This is an analytical, interventional, case series study de- 120 min for insulin and glucose measurements. Individ- signed as a single group clinical trial in pre-diabetic sub- uals with baseline fasting glucose at OGTT <99 mg/dL jects on a hypocaloric low glycemic index diet plus and >140 mg/dl and glucose levels <200 mg/dL 120 min metformin. The study lasted 16 weeks with monthly after the ingestion of oral glucose (impaired glucose toler- follow-up visits (total of 5 visits) to verify adhesion to ance) were selected [.
the dietary treatment, to adjust dietary prescription, and Values for the homeostasis model assessment of insu- to receive medication.
lin resistance (HOMA2-IR), HOMA β-cell function Metformin was kindly donated by Medley Laboratory, (HOMA2- % β), and HOMA insulin sensitivity (HOMA2- Brazil. The dose of 1 g/day metformin was divided into % S) were calculated using the computer software HOMA two doses (500 mg) taken at breakfast and dinner. The calculator v2.2.2 (University of Oxford; to determine insulin subjects were instructed to start the metformin treat- resistance, β-cell function, and insulin sensitivity, respect- ment with 500 mg per day and after 7 days increase to ively [The Cederholm index (CI) was calculated to 500 mg twice a day. In presence of any side effect they determine peripheral insulin sensitivity The calcula- were instructed to return to the single dose for 7 days tion, based on an appropriate analytical matrix and unified and to increase to twice a day subsequently. During the units, has been utilized in our previous studies in obese follow-up period no subjects reported any side effect.
subject Plasma glucose concentration was expressed Casulari et al. Diabetology & Metabolic Syndrome (2015) 7:65 in mmol/L and insulin concentration in pmol/L. The incre- calculated [(body weight (Kg) / height (m2)] and classi- mental area under the curve (AUC) for insulin and glucose fied according to parameters established by the World was calculated excluding any values below the baseline Health Organization Waist circumference was mea- using the trapezoidal method sured at the midline between the lowest rib and the iliaccrest with precision of 0.1 cm. Body fat percentage was Dietary and physical activity assessment calculated by using a tetrapolar electrical bioimpedance Dietary adherence and physical activity were assessed dur- measurement (Bioelectrical Impedance Analysis (BIA) ing the follow-up period according to the methodology Test–Quantum II RJL® Portable Body Fat Analyzer, described by the Dietary Reference Intakes (DRI) USA) All measurements and classifications were The subjects followed a hypocaloric low glycemic done according to standardized protocols always by the index diet. Dietary energy reduction was set between 25 same investigator.
and 30 % of the total energy expenditure, and the dietarymacronutrient percentages were based on the acceptable Biochemical analysis macronutrient distribution range Dietary energy Fasting state was verified using a glucometer (Roche was divided into food groups according to the Brazilian Diagnostics, Mannheim, Germany) in capillary blood food pyramid. A glycemic index table was created and samples. Blood samples drawn in the OGTT or post dexa- organized into food groups. At the beginning of the methasone tests were centrifuged and serum separated.
study, participants were instructed in the use of their Serum insulin levels were measured by chemiluminescence diet. The table was meant to help participants adhere to (Elecsys 2010, Roche Diagnostics, Mannheim, Germany), glucose by oxidasis-GOD/POD-automatized (Immulite The adherence to the treatment and the diet was 2000, DPC, Los Angeles, USA), cortisol by electrochemilu- checked in a monthly visit to the laboratory where a minescence (Elecsys 2010, Roche Diagnostics, Mannheim, follow-up questionnaire was administered and another Germany), total cholesterol, high density lipoprotein 30-day card of metformin pills was given to the subjects.
(HDL), and triacylglycerol by enzymatic colorimetric kits They were instructed to bring the used pill card for veri- (Labtest Diagnostica S.A., Belo Horizonte, Brazil). LDL- fication. Adherence to the diet was acknowledged in cholesterol and VLDL-cholesterol fractions were calcu- subjects who made 4–5 visits, did not ingest food that lated using the Friedwald equation .
gave them more than the proposed dietary energy andhad 4–6 meals per day. The dietary fiber intake was set- Glucocorticoid receptors tled to ≥ 70 % of the dietary reference intake of the DRI Blood for studying glucocorticoid receptors (GR) was Food intake was assessed by repeated 24-h recalls drawn by three Vacutainer ® tubes with EDTA. Blood in each visit. To ensure accuracy, a photograph of a food mononuclear cells (lymphocytes and monocytes) were portion guide was shown to all participants estimate the isolated by the protocol described by Ulmer and Flad consumed food portions. Each dietary recall was The cells extracted were stored in Eppendorf tubes reviewed in the presence of the participant to ensure its with a preserving solution and kept in a freezer at -80 °C accuracy and completeness. Food portions were con- until sent to the "Dipartimento di Scienze Farmacolo- verted into grams, and total energy intake and consump- giche Biomolecolari, Università di Milano, Sezione di tion of macronutrients and fiber were analyzed using the Biomedicina ed Endocrinologia" for analysis. For West- Nutrition Data System for Research software (version ern Blotting analysis the cellular lysates were resolved on 2011) (NDSR, University of Minnesota, Minneapolis, 7.5 % SDS-polyacrylamide gel electrophoresis (SDS- MN, USA), which includes typical Brazilian foods pre- PAGE), and transferred onto a nitrocellulose membrane.
pared using standardized recipes. Glycemic index of the GR protein was visualized with GR rabbit polyclonal anti- diets were calculated to be below 60.
body (M-20, Santa Cruz Biotechnology, Santa Cruz, Calif., The level of physical activity was assessed using a USA), diluted 1:100 . Samples from the last six partici- short-version of the international physical activity ques- pants – four women and two men – were analyzed.
tionnaire, and total energy expenditure was estimated byadding the appropriate physical activity level (PAL) to Statistical analysis the equations published by the DRI .
A correlation analysis between variables was conductedusing Pearson's correlation coefficient (two-tailed) test.
Anthropometric and body composition assessment All the characteristics measured, expressed as differences Body weight was assessed using an electronic platform from baseline, were compared after 16 weeks of inter- scale ranging from 0 to 150 kg and precision of 0.1 Kg; vention using the unpaired Wilcoxon test or Student t height was measured using a scientific stadiometer ran- test, according to the absence or presence of normality, ging from 0 to 210 cm and precision of 0.1 cm. BMI was respectively, and evaluated by the Kolmogorov-Smirnov Casulari et al. Diabetology & Metabolic Syndrome (2015) 7:65 test. All statistical analyses were done using the Statis- relevant positive correlation of insulin (r + 0.76; p = tical Analysis System software, version 9.1 (SAS Institute 0.08), HOMA-IR (r + 0.78; p = 0.07), HOMA2- % β (r + Inc., Cary, NC, USA), with statistical significance ≤ 0.05, 0.75; p = 0.09) and a negative correlation of HOMA2- % S (r - 0.75; p = 0.07) was observed with the glucocortic-oid receptors in basal conditions, even if the values did not reach the level of statistical significance.
Sixteen subjects were included. Of these, nine were men There were no significant correlations among cortisol and seven, women. Their average age was 34.6 SD 7 years and glucocorticoid receptor levels with body weight, (ranging from 19 to 49 years old), but the evaluation of waist circumference, body fat, total cholesterol, HDLc, glucocorticoid receptors was conducted in six patients– and LDLc (data not shown).
four men and two women. They were the last six partici-pants included in the study.
Table shows the level of serum cortisol in basal con- The epidemic rise of obesity and type-2 diabetes world- ditions and after suppression by dexamethasone, as well wide is partly due to the excessive intake of inadequate as glucocorticoid receptors in lymphocytes and mono- nutrients and little physical activity . Before the onset of cytes. Basal cortisol levels were not altered after treat- type-2 diabetes, predisposed individuals develop pre- ment (p = 0.24). A significant decrease occurred after the diabetic conditions that lead to the disease. The progres- use of dexamethasone before (p < 0.001) and after treat- sion to diabetes is weak for subjects with impaired fasting ment (p = 0.01). Cortisol levels after dexamethasone glucose, intermediate for those with impaired glucose tol- were higher after treatment (p = 0.02). Anthropometric erance and strong for those with both conditions [.
and laboratory analysis were not different from that of In this study, overweight or obese individuals with im- the whole group. The glucocorticoid receptor expression paired glucose tolerance were treated with metformin did not change after diet and metformin use as com- and a hypocaloric low glycemic index diet for four pared to baseline (p = 0.66).
months. The clinical results of the therapeutical associ- Table shows the correlation coefficient of selected ation of the diet and metformin in these patients were variables and basal cortisol level, post-dexamethasone previously published. The treatment produced a signifi- cortisol level and number of glucocorticoid receptors in cant decrease in body weight, BMI, waist circumference, lymphocytes and monocytes in basal conditions and and body fat over the follow-up period. No change in after 16 weeks of intervention. A positive correlation physical activity was observed during the intervention was observed between BMI and basal cortisol (r = 0.50; weeks. In addition, a significant reduction in CI, HOMA2- p = 0.05) after treatment. Glucocorticoid receptors levels % β, triglycerides, and VLDL levels was observed .
before treatment were strongly and positively correlated The homeostatic model assessment (HOMA) with BMI (r + 0.88; p = 0.02) and insulin AUC (r +0.94; used to quantify insulin resistance and beta-cell function p = 0.005). After intervention, there were no correlations indicates that the treatment has produced a reduction of with BMI (r -0.19; p = 0.71) and the correlation with in- the beta-cell function (HOMA2- % β), which was no sulin AUC became negative and non-statistically signifi- longer required to secrete a greater amount of insulin cant (r -0.72; p = 0.27). There were significant negative since insulin sensitivity was increased, as shown by a correlations between cortisol responses to dexametha- higher Cederholm Index (CI) sone and triglycerides (r - 0.68; p = 0.05) and VDLc (r – Cortisol secretion in these pre-diabetic subjects was 0.67; p = 0.05). These correlations were also found after tested evaluating plasma cortisol and utilizing the low treatment (r - 0.56; p = 0.04 and r - 0.55; p = 0.04). A doses of dexamethasone suppression test in an attempt Table 1 Cortisol levels in basal conditions and after suppression by dexamethasone; glucocorticoid receptors in lymphocytes andmonocytes at baseline and 16 weeks after a hypocaloric low glycemic index diet and metformin use in 16 subjects Basal cortisol (μg/dL) Post-dexamethasone cortisol (μg/dL) Glucocorticoid receptors d Data are expressed as mean and standard deviation (SD)a16 weeks vs. basal = paired Student t testbp < 0.001 basal cortisol vs. post-dexamethasone cortisol = paired Student t testcp = 0.01 basal cortisol vs. post-dexamethasone cortisol = Wilcoxon testdn = 6 subjects (4 male, 2 female) Casulari et al. Diabetology & Metabolic Syndrome (2015) 7:65 Table 2 Pearson correlation of selected variables and basal cortisol level, post-dexamethasone cortisol level and number ofglucocorticoid receptors in lymphocytes and monocytes in basal conditions and 16 weeks after a hypocaloric low glycemic indexdiet and metformin use in 16 subjects aAbbreviations: BMI = body mass index; AUC glucose = area under the glycaemic curve; AUC insulin = area under the insulinaemic curve; HOMA2-%β = homeostasismodel assessment β-cell function; HOMA2-IR = homeostasis model assessment insulin resistance; HOMA2-%S = homeostasis model assessment insulin sensitivity;VLDL-c = very low density lipoprotein cholesterolb6 subjects (4 male and 2 female) to detect subtle changes in the feedback of cortisol con- To the authors' knowledge a single paper is available in the trol in the hypothalamus and pituitary gland. Cortisol literature on the relationships between insulin secretion, levels and physiological cortisol suppression occurred in insulin resistance and corticosteroid receptors. In agree- response to low doses of dexamethasone, before and ment with the results of the study here presented, Islam after intervention, in agreement with previous investiga- et al. ] reported, in a study including 78 normo or tions conducted to verify normal and abnormal secretion slightly hyperglycemic subjects, that glucocorticoid recep- of this hormone We can infer from these re- tor concentration in leukocytes is significantly and posi- sults that cortisol secretion is preserved in obese individ- tively correlated with insulin resistance and BMI.
uals in pre-diabetic condition.
However, it is worth of note that there is a strong cor- The HPA axis activity in obese and type 1 diabetic pa- relation between corticosteroid receptors and insulin tients has been investigated in several studies with con- AUC, BMI and, to a minor extent, insulin levels and in- flicting results. In obese individuals cortisol levels have sulin resistance in the pre-treatment period, when the been reported to be normal while in other stud- insulin secretion was increased. After metformin and ies fasting and postprandial cortisol secretion in obese hypocaloric diet treatment, when insulin sensitivity patients has been described as lower than in lean sub- improved, this correlation disappears. The beneficial jects [In agreement with the findings of this study effect of this type of metformin and hypocaloric diet several authors have reported normal corti- treatment in improving insulin sensitivity was also de- sol suppression tests in response to low doses dexa- scribed in two previous articles by some of the au- methasone in obese individuals.
thors of our research group. In the first one, quoted In patients with type 2 diabetes the hypothalamic above, metformin and hypocaloric diet showed benefi- pituitary-adrenal (HPA) axis activity appears increased in cial effects in anthropometric and metabolic parame- most studies: elevation of ACTH [of basal ters of the same group of subjects with impaired glucose ] and suppressed serum cortisol (after dexamethasone tolerance In the second study, an increase in the test) [, and of late-night salivary cortisol levels ] levels of total and free testosterone was observed in sub- have been reported. In particular the presence of chronic jects with hypogonadotropic hypogonadism with meta- complications of type 2 diabetes (i.e., macroangiopathy, bolic syndrome .
retinopathy, and neuropathy) have been associated to with The mechanisms involved in metformin hypoglycemic HPA axis overactivity , –].
action in diabetic or glucose intolerant subjects are still The corticosteroid receptor density measured in a subset unknown –Metformin has a glucose lowering ef- of our patients in leukocytes does not show a significant fect related to reduction in hepatic gluconeogenesis. The variation between the pre and post-treatment conditions.
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Casulari LA, Caldas ADA, Motta LDC, Porto AL. Effects of metformin andshort-term lifestyle modifications on the improvement of male and take full advantage of:
hypogonadism associated with metabolic syndrome. Minerva Endocrinol.
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Acupuncture and Schizophrenia – Effect and Acceptability:Preliminary results of the first UK studyPatricia Ronan, Dominic Harbinson, Douglas MacInnes, Wendy Lewis, After several years of planning, we have just completed the intervention phase of a small, pre-clinical pilot study to explore the acceptability and effects of