Effect of diets supplemented with different conjugated linoleic acid (CLA) isomers on protein expression in C57/BL6 mice.

BACKGROUND: The individual genetic variations, as a response to diet, have recently caught the attention of several researchers. In addition, there is also a trend to assume food containing beneficial substances, or to supplement food with specific compounds. Among these, there is the conjugated linoleic acid (CLA), which has been demonstrated to reduce fat mass and to increase lean mass, even though its mechanism of action is still not known. We investigated the effect of CLA isomers (CLA c9,t11 and CLA t10,c12) on the proteomic profile of liver, adipose tissue, and muscle of mouse, with the aim of verifying the presence of a modification in fat and lean mass, and to explore the mechanism of action. METHODS: C57/BL6 mice were fed for 2 months with different diets: (1) standard chow, (2) CLA c9,t11 diet, (3) CLA t10,c11 diet, (4) CLA isomers mixture diet, and (5) linoleic acid diet. The proteomic profile of liver, white adipose tissue, and muscle was investigated. Statistical significance of the spots with an intensity higher than twofold in expression compared to the control was tested using student's t test (two-tail). RESULTS: We found that both isomers modulate the proteomic profiles of liver, adipose tissue, and muscle by different mechanisms of action. Liver steatosis is mostly due to the isomer CLA t10,c12, since it alters the expression of lipogenetic proteins; it acts also reducing the adipose tissue and increasing fatty acid oxidation in muscle. Conversely, CLA c9,t11 has no relevant effects on liver and adipose tissue, but acts mostly on muscle, where it enhances muscular cell differentiation. CONCLUSIONS: Administration of CLA in humans has to be carefully personalized, since even considering the presence of a species-specific effect, adverse effects might occur on long-term supplementation. Here we demonstrated that, in mouse, CLA is effective in reducing fat mass, but it also induces liver steatosis. The increase of lean mass is linked to an induction of cell proliferation, which, on long-term supplementation, might also lead to adverse effects.

Glycosylated serum protein levels assayed with highly sensitive immunoradiometric assay accurately reflect glycemic control of diabetic patients.

OBJECTIVE: To develop a sensitive and reliable immunoreadiometric assay to measure glycosylated lysine residues on serum proteins (GSP) and to evaluate its efficacy in monitoring glycemic control. RESEARCH DESIGN: The effect of acute and chronic in vitro and in vivo changes in glucose levels on GSP concentration was evaluated. GSP determinations from insulin-dependent diabetic (IDDM) patients, non-insulin-dependent diabetic (NIDDM) patients, and control subjects were correlated with other indices of glycemic control. RESULTS: The GSP levels were unaffected by acute glucose changes after food or intravenous glucose administration but increased during storage at -20 degrees C due to in vitro glycosylation by endogenous glucose. Immediate acidification of the serum prevented this, permitting long-term storage despite high ambient glucose levels. In randomly selected diabetic patients, 96% of GSP values were greater than the mean +3SD of nondiabetic control subjects. In diabetic patients, GSP levels correlated with mean plasma glucose concentrations (Kendall correlation statistics 0.47, P less than 0.001), fasting plasma glucose levels (Kendall statistics 0.42, P less than 0.001), and glycosylated hemoglobin (GHb, Kendall statistics 0.30, P less than 0.005). Induction of near-normal glycemia in poorly controlled NIDDM patients reduced GSP levels with a slope consistent with a half time of disappearance of 4.7 +/- 0.4 days. GSP levels remained elevated in 6 of 10 well-controlled NIDDM patients, despite normal GHb concentrations. Chronic hypoglycemic states, like pregnancy and hyperinsulinemic hypoglycemia, were associated with significantly low GSP levels. CONCLUSION: We describe a reproducible and sensitive immunoradiometric assay for GSP that closely reflects the degree of glycemic control in diabetic patients. Further studies are needed to determine whether this assay may be useful in screening for glucose intolerance or gestational diabetes.

Adrenomedullary hyperactivity in type I diabetic patients before and during continuous subcutaneous insulin treatment.

Urinary norepinephrine (NE) and epinephrine (E) excretion was measured at 4-h intervals for 2 consecutive days in nine type I diabetic patients with no signs of autonomic neuropathy before and after 3 weeks of glycemic control with continuous insulin infusion (CSII). Twenty-four-hour urinary E excretion was significantly higher in the diabetic patients than in normal subjects both before and after the period of CSII treatment [mean, 198.9 +/- 20.6 +/- SE and 127.8 +/- 24.4 vs, 46.6 +/- 9.8 nmol/day; P less than 0.05 for both]. The values in each of the 4-h periods before and in two of three of the periods after the 3-week period of CSII were significantly higher than those in normal subjects. Total urinary NE excretion was similar to that in the normal subjects at both times. The 24-h urinary NE/E ratio was significantly lower in diabetic patients even after they had achieved good metabolic control, compared with that in normal subjects (1.4 +/- 0.2 vs, 11.6 +/- 3.7; P less than 0.03). These data demonstrate hyperactivity of the adrenal medulla in type I diabetic patients, which is only partially reversed by a short period of glycemic control.

Effect of estradiol on the sympathoadrenal response to mental stress in normal men.

It has been shown that steroid hormones are able to influence the sympathoadrenal system activity. Therefore, we have investigated in a double blind cross-over study the effect of percutaneous estradiol administration (100 micrograms) on the sympathoadrenal and cardiovascular responses to mental arithmetic stress in 20 normal young males. The plasma estradiol level was 154 +/- 14 pmol/L during the estrogen session (ES) and 44 +/- 7 pmol/L during the placebo session (PL; P < 0.001). The mental stress induced a significant increase in systolic blood pressure during both the PL (F = 7.2; P < 0.001) and the ES (F = 4.8; P < 0.01); the peak obtained during PL was, however, higher than that during ES (128 +/- 2 vs. 122 +/- 3 mm Hg; P < 0.02). A significant increase in pulse rate was observed during PL (F = 4.2; P < 0.002), but not during ES (F = 2.6; P = 0.47), with the peak pulse rate being higher during mental stress in the PL than the ES (78 +/- 2 vs. 74 +/- 2 beats/min; P < 0.03). In response to the mental stress, plasma epinephrine increased significantly during PL (F = 3.2; P < 0.03), but not during ES (F = 1.1; P = 0.3). The stress-induced peak in plasma epinephrine during PL was higher than that during ES when expressed as the absolute value or the incremental peak (513 +/- 103 vs. 125 +/- 32 pmol/L; P < 0.005). The incremental peak in plasma norepinephrine obtained during PL was higher than that during ES (0.78 +/- 0.1 vs. 0.27 +/- 0.07 nmol/L; P < 0.02). Plasma free fatty acid, acetoacetate, and 3-hydroxybutyrate increased significantly from basal values during PL, but not during ES. These data show that mildly elevated levels of estradiol are able to influence the response of the adrenal medulla to mental stress in men.

Effect of 6-month gliclazide treatment on insulin release and sensitivity to endogenous insulin in NIDDM: role of initial continuous subcutaneous insulin infusion-induced normoglycemia.

In 10 obese, new-onset non-insulin-dependent diabetes mellitus (NIDDM) patients (group A), continuous subcutaneous insulin infusion (CSII) was used to induce normoglycemia during 14 days. Fasting blood glucose was 4.6 +/- 0.2 mmol/L and mean daily blood glucose 5.8 +/- 0.2 mmol/L at the end of the CSII period. This excellent glycemic control was obtained with 35 +/- 4.8 U of insulin per day, corresponding to 0.47 +/- 0.06 U/kg/24 hours. Endogenous insulin production was markedly suppressed, since urinary C-peptide was reduced from 18.5 +/- 0.12 to 7.9 +/- 0.25 nmol/24 hours. Gliclazide was given to group A following CSII, and to five obese NIDDM patients (group B) in their habitual hyperglycemic state. Gliclazide maintained in group A, and induced in group B, excellent metabolic control. This was accompanied by the appearance of a small first-phase insulin response to intravenous glucose, and significant increases in the mean-daily-insulin to mean-daily-blood-glucose ratio, as well as in the 24-hour urinary C-peptide-to-glucose ratio. The gliclazide effects tended to be more pronounced in group A. No significant effect was seen on efficacy of endogenous insulin (slope of disappearance of blood glucose divided by insulin levels). During 6 months of gliclazide treatment, excellent glycemic control was maintained in all patients. This was paralleled by unchanged stimulation by gliclazide of first-phase insulin response to glucose, and augmented mean 48-hour insulin-to-glucose and urinary C-peptide-to-glucose ratios. No change in the ratio of glucose disposal to endogenous insulin was noted. We conclude that physiologic insulin replacement may induce normoglycemia in NIDDM, indicating that insulin resistance is not of clinical significance; gliclazide has a beta-cell-stimulating action that is maintained quantitatively unchanged for at least 6 months; the therapeutic effect of gliclazide in NIDDM seems to be mainly, if not exclusively, the result of its beta-cytotrophic action. Initial normoglycemia, induced here by CSII, may have a lasting enhancing effect on the gliclazide action.

Plasma catecholamines after thyrotropin-releasing hormone administration in hypothyroid patients before and during therapy.

In order to investigate sympathoadrenal activity in hypothyroidism we studied the cardiovascular and catecholamine responses to thyrotropin-releasing hormone (TRH) infusion in nine hypothyroid patients before and during adequate therapy and in seven healthy subjects. We evaluated mean arterial pressure, heart rate, plasma epinephrine and norepinephrine levels after TRH administration (200 micrograms iv) in the three groups. Mean arterial pressure, heart rate and plasma epinephrine levels were not different in the three groups and did not change after TRH administration. Hypothyroid subjects showed increased plasma norepinephrine levels (1.48 +/- 0.15 nmol/l), which were reduced after euthyroidism was reached (0.84 +/- 0.11 nmol/l) (p < 0.01). An exaggerated response of norepinephrine to TRH was observed in hypothyroid patients before therapy (incremental peak (IP) = 0.59 +/- 0.13 nmol/l) but not in hypothyroid patients during therapy (IP = 0.19 +/- 0.02 nmol/l p < 0.02) or in the control group (IP = 0.15 +/- 0.04 nmol/l; p < 0.05). This study indicated that TRH administration is able to influence the sympathetic activity during hypothyroidism in humans.

Effect of 6 months' gliclazide treatment on insulin release and sensitivity to endogenous insulin in NIDDM: role of initial CSII-induced normoglycemia.

In 10 obese, newly diagnosed non-insulin-dependent diabetes mellitus (NIDDM) patients (group A) continuous subcutaneous insulin infusion (CSII) was used to induce normoglycemia over a period of 14 days. Fasting blood glucose was 4.61 +/- 0.22 mmol/l and mean daily blood glucose 5.83 +/- 0.27 mmol/l at the end of the CSII period. This excellent glycemic control was obtained with 35 +/- 4.8 U insulin per day, corresponding to 0.47 +/- 0.06 U/kg/24 h. Endogenous insulin production was markedly suppressed, since urinary C-peptide was reduced from 56 +/- 0.35 to 24 +/- 0.76 micrograms/24 h. Thus, physiological insulin replacement induced normoglycemia in NIDDM, indicating that insulin resistance is not clinically important. Gliclazide was given to group A following CSII and to 5 obese NIDDM patients (group B) in their habitual hyperglycemic state. Gliclazide maintained in group A and induced in group B excellent metabolic control. This was accompanied by the appearance of a small first-phase insulin response to iv glucose and by significant increases in the mean daily insulin to mean daily blood glucose ratio and in the 24-h urinary C-peptide to glucose ratio. The gliclazide effects tended to be more pronounced in group A. No significant effect was seen on sensitivity to endogenous insulin (slope of disappearance of blood glucose as function of insulin response to glucose infusion). During the 6 months of gliclazide treatment, excellent glycemic control was obtained in all patients. This was paralleled by unchanged stimulation by gliclazide of first-phase insulin response to glucose as well as mean by 48-h insulin to glucose and urinary C-peptide to glucose ratios. Again, sensitivity to endogenous insulin was not augmented. We conclude that gliclazide has a beta-cell-stimulating action which is maintained quantitatively unchanged for at least 6 months. The therapeutic effect of gliclazide in NIDDM seems to be mainly, if not exclusively, the result of its beta-cytotrophic action. Initial normoglycemia, induced here by CSII, may have a lasting enhancing effect on gliclazide action.

Conjugated linoleic acid isomers modulate protein expression profile in rat hepatocytes.

Conjugated linoleic acid (CLA) is a polyunsaturated fatty acid, which has been recently proven to be effective in reducing body fat mass, but brings as a side effect, the liver enlargement due to an increased lipid content. The in vivo lipogenic activity has been suggested to be due to the reduction in fat mass and to the consequent metabolism of blood glucose to fatty acid in the liver rather than in the adipose tissue. We investigated the ability of CLA to directly induce steatosis by modulating the expression pattern of hepatic proteins involved in lipid metabolism. To avoid interferences derived from CLA metabolism by other tissues, we used the in vitro model of freshly isolated rat hepatocytes incubated in the presence of different CLA isomers. The direct effect of CLA on lipid accumulation in hepatocytes was demonstrated by the altered expression pattern of several proteins involved in lipid metabolism, as assessed by two-dimensional gel electrophoresis and confirmed by Western blotting analysis. The CLA isomer c9,t11 was most effective in modulating the protein expression profile.

High urinary excretion of adrenaline in insulin dependent diabetic subjects.

In view of the hyperglycemic and ketogenic actions of catecholamines, studies on the adrenergic pattern in diabetic patients are relevant to the management of diabetes. We have studied urinary adrenaline and noradrenaline excretion in 4-hour collections in 16 type I diabetic male patients without signs of autonomic or peripheral neuropathy and 11 age-weight matched controls. In diabetic patients adrenaline levels were higher than control subjects (26.8 +/- 3.9 vs 10.7 +/- 3.0 nmol/4h; p < 0.003) but did not differ in respect of noradrenaline (62.6 +/- 6.8 vs 59.6 +/- 10.8 nmol/4h) and creatinine excretion. This finding demonstrates a hyperactivity of the adrenal medulla in diabetic patients without concomitant elevations of noradrenaline. This occurred in absence of hypoglycemia and seems to be a common feature of type I diabetics without complications. Since these levels of adrenaline are sufficient to stimulate both lypolysis and glycogenolysis a previous characterization of adrenomedullary activity may help to better define insulin demands in diabetic patients.

Systolic time intervals in isolated septal defects before and after corrective surgery. II. Ventricular septal defect.

Systolic time intervals (STI) were analyzed in 34 patients with isolated ventricular septal defect (VSD) and undirectional left-to-right shunt. 14 of the patients who underwent corrective surgery were followed-up for at least 10 years. The measurements were obtained from simultaneous high speed photographic recordings of electrocardiogram, external carotid pulse and phonocardiogram. Before the operation the Q-I sound interval and, consequently, the pre-ejection period were significantly prolonged and the left ventricular ejection time significantly abbreviated, the degree of abbreviation relating with the magnitude of the shunt. The above deviations persisted after corrective surgery, and only several years after the operation the parameters studied became normal. It is concluded that the most likely explanation for these abnormalities is the depressed contractility of the left ventricle, secondary to the long-standing volume overload, which tends to persist after corrective surgery.

Systolic time intervals in isolated septal defects before and after corrective surgery in female patients. I. Atrial septal defect.

Systolic time intervals (STI) were evaluated in 19 female patients with uncomplicated ostium secundum atrial septal defect (ASD), before, shortly after (within 2 months) and a long time after the corrective surgery (mean 13.2 years). The measurements were obtained from simultaneous high speed photographic recordings of electrocardiogram, external carotid pulse and phonocardiogram. Before the operation, a significant prolongation of Q-I heart sound interval was detected, together with a less prominent but statistically significant shortening of the left ventricular ejection time; the above alterations were not correlated with the magnitude of the shunt and disappeared shortly after corrective surgery. The STI were still normal a long time after surgical closure of ASD. In accordance with hemodynamic studies reported by other authors, our results confirm that the deviations of STI observed in ASD are due to a reduced diastolic filling of the left ventricle secondary to an apparent decreased distensibility.

Effects of androgen treatment in impotent men with normal and low levels of free testosterone.

The relation between sexual function and serum free testosterone (fT) levels, which represent the active fraction of circulating testosterone, was evaluated. Two groups of impotent male subjects with mild hypogonadism were treated with oral testosterone undecanoate (TU); these men presented with tT/luteinizing hormone (LH) ratio and tT levels at the lower limits of normal. The first group had serum fT below 6.6 ng/ml, considered the lower normal value, according to our laboratory method, whereas the second group had normal fT limits. Administration of TU improved sexual function only in impotent men with low fT levels, but not in subjects with normal fT levels, even though the tT levels and the tT/LH ratio of the two groups were not significantly different. The results of our study suggest the presence of a minimum serum fT threshold, lying near the lower normal range, which determines the male sexual function. Moreover, serum fT levels were a more sensitive index than tT for identifying impotent men who can be successfully treated with androgens.

Chronobiology of catecholamine excretion in normal and diabetic men.

The adrenomedullary response to stimuli is often elevated in poorly controlled insulin dependent diabetic patients, and it is controversial whether the adrenomedullary hyperactivity induces the suppression of the circadian rhythm of catecholamines. We have studied the urinary excretion of catecholamines in 11 diabetic patients during 48 h in 4-h collections. Eleven age and weight matched normal subjects served as controls. A circadian rhythm was detected for adrenaline and noradrenaline excretion both in normal and diabetic subjects, with the highest value for both catecholamines in the early afternoon. The mean daily adrenaline levels were significantly higher in diabetic than in control subjects (p less than 0.05). The dopamine excretion was correlated with noradrenaline excretion in normal subjects but did not show a definite circadian rhythm. We conclude that the adrenomedullary hyperactivity does not affect the rhythmic fluctuations of adrenaline and noradrenaline. The dopamine excretion does not show circadian variations and this probably reflects the absence of a single controlling oscillator.

Daily variations in catecholamine excretion are not influenced by very low calorie diet in obese women.

The increase in metabolic efficiency during energy restriction seems to be an established phenomenon in obese patients. The sympathoadrenal system is involved in the control of energy expenditure and the catecholamine responses to stimuli vary during the day. We therefore studied the circadian pattern of urinary catecholamine excretion during 4-h collections for two consecutive days in a group of 20 obese female patients during and after a very low calorie diet (500 kcal/die). The diet period induced a significant weight loss in all the patients studied (99.1 +/- 3.7 vs 92.5 +/- 4.1 Kg; p < 0.01). The mean daily excretion of epinephrine did not change after 24 days of diet restriction when compared with the value obtained at the 4th day (12.0 +/- 2.5 vs 10.3 +/- 2.2 nmol/4 h respectively; p = NS) while a slight decrease was observed in the mean daily excretion of norepinephrine (52.5 +/- 8.7 vs 66.6 +/- 9.3 nmol/4 h respectively; p = NS). A circadian rhythm was detected for epinephrine and norepinephrine excretion both during and after very low calorie diet. No significant changes were found in the chronobiological characteristics of epinephrine and norepinephrine with the peak of excretion in the afternoon both during (epinephrine: 16:30 h; norepinephrine: 16:45 h) and after the diet (epinephrine: 17:35 h; norepinephrine: 18:00 h). It seems doubtful that alterations in the chronobiological pattern of catecholamines play a role in the metabolic adaptation occurring during very low calorie diet in obese females.

Changes in the bioactivity to immunoreactivity ratio of circulating luteinizing hormone in impotent men treated with testosterone undecanoate.

Testosterone undecanoate was administered orally (80 mg twice daily) for 30 days to 10 impotent men with mild Leydig cell failure, age 28 to 42 years. Placebo was administered for 30 days both before and at the end of testosterone undecanoate therapy. Serum levels of bioactive LH, immunoreactive LH and testosterone were determined in basal conditions (day zero), 30 days after the first placebo administration, at the 15th and 30th day of testosterone undecanoate therapy, and at the end of the second treatment with placebo (90th day). Bioactive LH was measured by a sensitive and specific in vitro bioassay based on testosterone production by mechanically dispersed mouse Leydig cell preparations. Immunoreactive LH and testosterone were determined by a double-antibody RIA technique. The results were compared with those obtained in 30 untreated normal young men. In the basal state, serum concentrations of immunoreactive LH were significantly higher in the patients (P less than 0.02) than in control subjects, whereas testosterone levels were significantly lower (P less than 0.001) in the impotent men. In contrast, bioactive LH levels and the bioactive LH to immunoreactive LH ratios were similar in the two groups. In the patients, at the 15th day of treatment with testosterone undecanoate, serum levels of testosterone and bioactive LH were significantly higher (P less than 0.01) than basal values, whereas immunoreactive LH concentrations showed no significant changes. Consequently, the bioactive LH to immunoreactive LH ratios rose significantly (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Very low calorie diet induces opposite effects on sympathetic nervous system and adrenomedullary responses.

The effect of very low calorie diet (VLCD) on catecholamine excretion during 24 h was examined in nine obese men, with normal blood pressure, maintained on low sodium balance, after 3 days and 14 days of the diet period. A significant decrease in the total daily noradrenaline (NA) excretion was observed at the end of the diet (P less than 0.05). The daily excretion of adrenaline (A) increased (P less than 0.05) with a fall in the NA/A ratio (5.1 +/- 2.6 vs 12.5 +/- 3.7; P less than 0.03). The results demonstrate that the sympathetic nervous system is influenced to a greater extent by caloric intake than sodium homeostasis and that the VLCD exerts different effects on SNS and adrenomedullary secretion.