Riccardo Giorgino

Fat depot-related differences in gene expression, adiponectin secretion, and insulin action and signalling in human adipocytes differentiated in vitro from precursor stromal cells

Aim/hypothesisThe distinct metabolic properties of visceral and subcutaneous adipocytes may be due to inherent characteristics of the cells that are resident in each fat depot. To test this hypothesis, human adipocytes were differentiated in vitro from precursor stromal cells obtained from visceral and subcutaneous fat depots and analysed for genetic, biochemical and metabolic endpoints.MethodsStromal cells were isolated from adipose tissue depots of nondiabetic individuals. mRNA levels of adipocyte-specific proteins were determined by real-time RT-PCR. Insulin signalling was evaluated by immunoblotting with specific antibodies. Glucose transport was measured by a 2-deoxy-glucose uptake assay. Adiponectin secretion in the adipocyte-conditioned medium was determined by a specific RIA.ResultsWith cell differentiation, mRNA levels of PPARG, C/EBPα (also known as CEBPA), AP2 (also known as GTF3A), GLUT4 (also known as SLC2A4) were markedly upregulated, whereas GLUT1 (also known as SLC2A1) mRNA did not change. However, expression of C/EBPα, AP2 and adiponectin was higher in subcutaneous than in visceral adipocytes. By contrast, adiponectin was secreted at threefold higher rates by visceral than by subcutaneous adipocytes while visceral adipocytes also showed two- to threefold higher insulin-stimulated glucose uptake. Insulin-induced phosphorylation of the insulin receptor, IRS proteins, Akt and extracellular signal-regulated kinase-1/2 was more rapid and tended to decrease at earlier time-points in visceral than in subcutaneous adipocytes.Conclusions/interpretationSubcutaneous and visceral adipocytes, also when differentiated in vitro from precursor stromal cells, retain differences in gene expression, adiponectin secretion, and insulin action and signalling. Thus, the precursor cells that reside in the visceral and subcutaneous fat depots may already possess inherent and specific metabolic characteristics that will be expressed upon completion of the differentiation programme.

Gender differences in serum leptin in obese people: relationships with testosterone, body fat distribution and insulin sensitivity

Testosterone levels are decreased in obese men but increased in obese women. The interplay between gonadal steroids and leptin is, at present, far from being elucidated. This study was carried out to investigate the relationship between serum leptin, plasma insulin, insulin sensitivity and free testosterone in 46 men (29 obese and 17 lean) and 65 premenopausal women (42 obese and 23 lean). In all subjects, anthropometric parameters and serum levels of insulin, leptin, free testosterone (T), dehydroepiandrosterone sulphate and sex hormone‐binding globulin were measured. An oral glucose tolerance test (OGTT) and an insulin tolerance test were also performed to determine the insulin sensitivity index. Our results show a significant difference in serum leptin between lean and obese men (3.19 ± 0.71 vs. 20.28 ± 0.26 ng mL−1; P < 0.0005) as well as between lean and obese women (10.78 ± 2.14 vs. 34.79 ± 2.26 ng mL−1; P < 0.00001). Basal T concentration in the obese men was significantly lower than in the control group (18.6 ± 1.3 vs. 23.3 ± 1.4 ng L−1; P < 0.01), whereas in the obese women it was significantly higher than in the control group (2.0 ± 0.2 vs. 1.3 ± 0.1 ng L−1; P < 0.05). When multiple linear regression was performed without body mass index (BMI) in the statistical model, leptin was correlated with basal insulin (P < 0.0001), insulin sensitivity (P < 0.0001) and T (P < 0.0001) in both men and women. When BMI was included in the model as an independent variable, leptin was significantly correlated only with BMI (P < 0.0001), the degree of insulin resistance (P < 0.05) and T (only in men, P < 0.05). This study confirms that serum leptin is strongly correlated with the degree of obesity and female sex. The negative correlation between leptin and T in men, independent of BMI, is consistent with the hypothesis that T may possess an inhibitory effect on adipocyte ob gene transcription.

Up-regulation of androgen receptor binding in male rat fat pad adipose precursor cells exposed to testosterone: Study in a whole cell assay system

Binding of androgens to adipocytes has previously been evaluated using cytosol fractions without taking into account nuclear binding, although the latter is suggested to be close to the physiological site of action. In the present study, performed in differentiated fat pad adipose precursor cells, we describe a simple, reliable and reproducible androgen binding assay in a system with intact cells. Tritiated and unlabeled methyltrienolone (R1881) were used to define specific and unspecific androgen binding. Triamcinolone acetonide was added to prevent the binding of R1881 to other types of receptors. Differentiated adipose precursor cells contain a homogeneous class of high affinity androgen binding sites, and binding is saturable and reversible. Binding apparently occurs at one site, with a Kd in the range of physiological androgen concentration (about 4 nM). Competition studies indicate that the receptor is specific for R1881, testosterone and dihydrotestosterone, which have approximately the same affinity, while progesterone, estradiol and dexamethasone show much lower affinity. Androgen binding was markedly enhanced after cellular exposure to R1881 and testosterone but not dihydrotestosterone, and this increase was dependent on protein synthesis, suggesting the formation of new receptors by these androgens. In conclusion, fully differentiated adipocytes contain a specific, high affinity receptor, the density of which is dependent on androgens.

Inhibitory Effect of Obesity on Gonadotropin, Estradiol, and Inhibin B Levels in Fertile Women

Objective: To examine whether obesity and insulin resistance have an independent effect on the gonadotropin, estradiol, and inhibin B serum levels and follicle count in the early follicular phase of fertile women with a wide range of BMI and without signs of hyperandrogenism.

Lower androgenicity is associated with higher plasma levels of prothrombotic factors irrespective of age, obesity, body fat distribution, and related metabolic parameters in men

The purpose of this study was to examine the relationships between androgenic status and plasma levels of both prothrombotic and antithrombotic factors in men, irrespective of obesity, body fat distribution, and metabolic parameters. Sixty-four apparently healthy men, 40 with a body mass index (BMI) greater than 25 kg/m2 (overweight and obese [OO]) and 24 non-obese controls with a BMI less than 25, were selected and evaluated for (1) plasma concentrations of plasminogen activator inhibitor-1 (PAI-1) antigen, PAI-1 activity, fibrinogen, von Willebrand factor (vWF) antigen, vWF activity, and factor VII (FVII) as the prothrombotic factors; (2) plasma levels of tissue plasminogen activator (TPA) antigen, protein C, and antithrombin III as the antithrombotic factors; (3) fasting plasma concentrations of insulin and glucose and the lipid pattern (triglycerides [TG] and total and high-density lipoprotein [HDL] cholesterol) as the metabolic parameters; and (4) free testosterone (FT), dehydroepiandrosterone sulfate (DHEAS), and sex hormone-binding globulin (SHBG) serum levels as the parameters of androgenicity. Body fat distribution was evaluated by the waist to hip ratio (WHR). In OO and non-obese subjects taken together, plasma levels of PAI-1 antigen, fibrinogen, and FVII were inversely associated with FT (r = .255, P < .05, r = -3.14, P < .05, and r = -.278, P < .05, respectively), and the negative relationships of both fibrinogen and FVII with FT were maintained after stepwise multiple regression analysis. Plasma concentrations of PAI-1 antigen and PAI-1 activity were also negatively correlated with SHBG (r = -.315, P < .05 and r = -.362, P < .01, respectively), and these associations held irrespective of the other parameters investigated. None of the antithrombotic and fibrinolytic factors were independently related to serum androgen levels. Subjects with a BMI higher than 25 kg/m2 had higher plasma concentrations of PAI-1 antigen, PAI-1 activity, and fibrinogen as compared with non-obese controls (P < .001, P < .001, and P < .01, respectively). In addition, in OO and control subjects as a whole, multiple stepwise regression analysis showed that the associations of BMI with PAI-1 activity, fibrinogen, vWF antigen, and vWF activity were independent of any other metabolic and hormonal parameters. Plasma concentrations of PAI-1 antigen, PAI-1 activity, and fibrinogen were also directly correlated with WHR in all subjects taken together, irrespective of the other parameters investigated. Evaluation of antithrombotic factors showed that OO subjects had higher TPA plasma concentrations than non-obese controls (P < .001), whereas protein C and antithrombin III did not differ in the two groups. TPA was also directly correlated with BMI (r = .415, P < .001) and WHR (r = .393, P < .001) in all subjects. The results of this study indicate that (1) men with lower FT serum levels have higher fibrinogen and FVII plasma concentrations, and those with lower SHBG serum levels also have higher levels of PAI-1 antigen and activity; (2) irrespective of other factors, obesity per se may account for higher concentrations of PAI-1, fibrinogen, and vWF; (3) plasma levels of PAI-1 (antigen and activity) and fibrinogen correlate independently with WHR; and (4) among the investigated antithrombotic factors (TPA antigen, protein C, antithrombin III), only TPA antigen plasma concentrations are higher in men with abdominal obesity. Thus, because of the increase in several prothrombotic factors, men with central obesity, particularly those with lower androgenicity, seem to be at greater risk for coronary heart disease (CHD). Apparently, this risk is not counteracted by a parallel increase in plasma concentrations of antithrombotic factors.

Low dehydroepiandrosterone circulating levels in premenopausal obese women with very high body mass index

Dehydroepiandrosterone (DHEA) has an anti-obesity effect in rodents and reduces body fat in normal men. Therefore, the plasma levels of DHEA were evaluated in nine premenopausal healthy women and in 13 menstrually active nondiabetic obese women, including patients (n = 6) with body mass index (BMI) over 40. In the obese group, a significant inverse correlation between DHEA levels and BMI was found. These results suggest that patients with severe obesity are unable to increase the DHEA adrenal production rate in order to parallel the increase in the hormone metabolic clearance rate (due to enlargement of body fat mass per se). The deficiency of this mechanism might itself contribute to the progressive fat accumulation in severe obesity.

Urinary albumin excretion is independently associated with C-reactive protein levels in overweight and obese nondiabetic premenopausal women.

Abstract. Pannacciulli N, Cantatore FP, Minenna A, Bellacicco M, Giorgino R, De Pergola G (Section of Internal Medicine, Endocrinology and Metabolic Diseases and Section of Rheumatology, University of Bari, Bari, Italy). Urinary albumin excretion is independently associated with C‐reactive protein levels in overweight and obese nondiabetic premenopausal women. J Intern Med 2001; 250: 502–507.

High Systolic Blood Pressure Increases Prevalence and Severity Of Retinopathy in NIDDM Patients

OBJECTIVE To determine whether the severity of retinopathy is higher in a group of NIDDM patients with sBP ≥ 140 mmHg compared with NIDDM patients with sBP < 140 mmHg. RESEARCH DESIGN AND METHODS Ophthalmoscopy and FAG were conducted among a group of NIDDM patients with either a sBP above (n = 54) or below (n = 55) 140 mmHg. The groups were matched according to diabetes duration, metabolic control (HbA1c), and AER. RESULTS Patients with sBP > 140 mmHg had a higher prevalence of retinopathy, as established according to a rating scale (4.9 ± 3.8 vs. 3.2 ± 3.3, P < 0.02); furthermore, their BMI values were higher (28.1 ± 4.5 vs. 24.9 ± 4.1 kg/m2, P < 0.001). The group of normotensive subjects showed the highest rate of low grading (0–2) values. However, the highest prevalence rates of 8–10 grading values (proliferative retinopathy) were found in the hypertensive group. CONCLUSIONS These data suggest that sBP values ≥ 140 mmHg favor the onset of retinopathy in NIDDM patients during their 1st 10 yr of disease.

Relation between sex hormones and serum lipoprotein and lipoprotein(a) concentrations in premenopausal obese women.

Lipoprotein(a) (Lp[a]) is generally considered to be a risk factor for the development of cardiovascular disease, but little is known about the possible influence of obesity on the circulating levels of this lipoprotein. The present study was undertaken to examine this aspect in 136 menstrually active women by comparing the serum concentrations of Lp(a) between 72 obese and 64 age-matched nonobese women. Since an adverse effect of androgens and a protective effect of estrogens have been described for plasma lipoprotein profiles in obese women, the relation between the circulating levels of Lp(a) and those of these other hormones was also investigated in obese patients. In addition, other lipoproteins, anthropometric parameters (body mass index and waist-to-hip ratio), and insulin were evaluated. The levels of Lp(a) were not significantly different (Mann-Whitney U test chi 2, 3.59; p = 0.0582 [NS]) between obese (rank sum, 5,367) and control (rank sum, 3,949) women; in addition, the percentage of patients with high Lp(a) levels (cutoff defined at 30 mg/dL) did not differ between the two groups (obese women, 30%; control, 21.8%; chi 2, 0.90; two-sided p = 0.341 [NS]). Moreover, no correlation was found between Lp(a) and body mass index. Lastly, when the Lp(a) prevalence odds ratio for obesity was examined by adjusting the levels of this lipoprotein for age, triglycerides, total cholesterol, and high density lipoprotein cholesterol, the probability value (0.88) was far from significant. In obese women, no correlation was found between the logarithmically transformed Lp(a) concentrations and all the other variables evaluated in the study. In conclusion, the present study shows that the circulating levels of Lp(a) are not influenced by body weight and cardiovascular risk factors commonly associated with obesity, such as enhanced androgenic activity, hyperinsulinemia, adverse lipoprotein profile, and abdominal fat accumulation.

Influence of subclinical hypothyroidism and T4 treatment on the prevalence and severity of obstructive sleep apnoea syndrome (OSAS)

Background: Obstructive sleep apnoea (OSA) and subclinical hypothyroidism are relatively frequent disorders that may be causally linked. However, discordant results exist on the prevalence and severity of OSA in subclinical hypothyroidism. The aim of this study was to compare the prevalence and severity of sleep-dis-ordered breathing in individuals with or without subclinical hypothyroidism, and to investigate the possible effect of levothyroxine treatment on these patients. Patients and Methods: One hundred and eight subjects were consecutively enrolled and divided in 3 groups, according to the TSH levels and levothyroxine therapy. The first group (Group A) was represented by 63 subjects with normal TSH and thyroid function. The other two groups included patients affected by subclinical hypothyroidism; one group (Group B) treated with levothyroxine, while the other group (Group C) was never treated with levothyroxine. Anthropometric, respiratory and polysomnographic data were evaluated in all individuals. Results: The percentage of OSA, neck circumference, and body mass index (BMI) were not statistically different among the 3 groups. Respiratory disturbance index (RDI) as well as the percentage of the total number of events (apnoea-hypopnoea) by total sleep time (TST) with <90% oxyhemoglobin saturation (TSTSaO2 <90%) were not different among the groups. When we observed OSA patients, the only significant difference between groups B and C was represented by the Epworth Sleepiness Scale (ESS) (p=0.005). Conclusion: This study shows that subclinical hypothyroidism and treatment with levothyroxine do not influence the prevalence and severity of OSA, while sleep propensity is increased by untreated subclinical hypothyroidism.