1 Consultant Obstetrician and Gynaecologist, Department of Obstetrics and Gynaecology, Dryburn Hospital, Durham, 2 Consultant Gynaecologist, Centre for Reproductive Medicine, Royal Victoria Infirmary, Newcastle upon Tyne and 3 Professor of Metabolic Medicine, Department of Medicine, University of Newcastle upon Tyne, UK
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Abstract |
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Key words: hypogonadism/hypogonadotrophic/insulin action
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Introduction |
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Patients with hypogonadotrophic hypogonadism (HH) are also chronically anovulatory, albeit with a completely different sex steroid and gonadotrophin profile compared with PCOS. If the insulin resistance in PCOS is indeed a reflection of anovulation then patients with HH should also have a reduction in insulin sensitivity. The present study was therefore designed to investigate the hypothesis that patients with HH are also insulin resistant. The aim was to study insulin sensitivity of adipose tissue in amenorrhoeic patients with HH.
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Materials and methods |
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Methods
Subjects and controls were studied between 0800 and 0900 h after an overnight fast. Using an aseptic technique an incision was made (12 cm) just below the pubic hair line after s.c. infiltration with 1% lignocaine. Between 36 g of s.c. fat was obtained from the lower abdominal wall using dissecting forceps and a scalpel with a number 11 blade. The adipose tissue was then transported to the laboratory in glucose/saline (5 mmol/l glucose/154 mmol/l NaCl) with HEPES 10 mmol/l at 37°C. The adipocyte isolation techniques (Pedersen et al., 1981) were used. The adipose tissue was finely chopped and incubated for 90 min at 37°C in a HEPES buffer (pH 7.4), containing human serum albumin (25 g/l) and collagenase (0.5 g/l). The isolated adipocytes were then washed with a HEPES buffer, containing human serum albumin (50 g/l) as previously described (Taylor et al., 1985
). Tissues and cells were suspended in a HEPES buffer containing the following substance concentrations in mmol/l: NaCl 135, KCl 4.8, MgSO4 1.7, CaCl2 0.5, NaH2PO4 0.2, Na2HPO4 1.0. The pH was adjusted to 7.4 at 37°C. Human serum albumin was present at 50 mg/ml and glucose at 5 mmol/l except for the glucose transport experiments where it was essential that glucose free buffer was used. Cell number and total cell surface area/incubation tube were derived by measuring the diameter of 100 cells and calculating individual cell volume and cell surface area. The mean cell volume and surface area were calculated from the known lipocrit of the cell suspension (Pedersen et al., 1981
). Venous blood was taken on the day of biopsy for oestradiol, LH, FSH, testosterone, androstenedione, fasting glucose, fasting insulin, intermediate metabolite and non-esterified fatty acids estimation prior to commencing the in-vitro studies of insulin sensitivity. An index of in-vivo insulin sensitivity was derived using the homeostasis model assessment method (HOMA) (Matthews et al., 1985
).
Glucose transport
Glucose uptake was measured using the method described by Pedersen and Gliemann (1981). Glucose uptake was initiated with direct injection of 14 µl of 3-O-methylglucose (final concentration of 43.3 µmol/l) and the reaction was stopped at 5 s by adding 3 ml 154 mmol/l NaCl containing 0.3 mmol/l of phloretin and 0.2% v/v of ethanol. Silicone oil was layered on the surface and the tubes were spun within 2 min at 3000 g for 90 s. The cell pellets were harvested with a disposable plastic pipette tip and placed in a vial containing 5 ml of scintillation fluid. The trapped extracellular radioactivity was measured by adding 3 ml of saline/phloretin solution before the addition of the glucose and this value was subtracted from all observed values. Glucose transport was expressed as pmol 3-O-methylglucose/10 cm2 cell membrane.
Adipocyte insulin binding
Insulin binding to adipocytes was measured as previously described (Pedersen et al., 1981) using A14 labelled mono-[125I] insulin (final concentration of 0.710 pmol/l) and insulin (final concentration 1 pmol/l to 100 000 pmol/l) in duplicate incubation. Specific binding was calculated by subtracting the binding observed in the presence of 1.3x105 mol/l insulin from the total binding for each insulin concentration. The mean non-specific binding was 5.9% of total cell bound insulin. Binding was expressed as % specific binding/10 cm2 adipocyte surface area. Previous work in this laboratory has demonstrated specific insulin binding to adipocytes from a group of normal women to be 1.78 ± 0.18% (Marsden et al., 1994
).
Lipolysis inhibition
Lipolysis was measured by incubating 250 µl of 10% cell suspension with either 250 µl of buffer (basal rate) or 200 µl buffer and 50 µl noradrenaline (stimulated rate) or 150 µl buffer, 50 µl noradrenaline and varying concentrations of insulin (1014 to 1010 mol/l) for 90 min at 37°C in a shaking waterbath. The medium did not contain caffeine as previously described (Pedersen and Hjollund, 1982) and this is likely to account for the observed exquisite sensitivity to insulin. The incubation was terminated with 2 ml of silicone oil and the tubes were centrifuged at 3000 g for 5 min. The silicone oil and the cells were then aspirated with a glass pipette and the infranatant was then stored at 40°C for subsequent assay of glycerol concentration. For glycerol analysis a perchloric acid (PCA) extract was prepared and glycerol was measured using an enzymatic fluorometric continuous-flow assay (Lloyd et al., 1978
). Lipolysis was expressed as nmol glycerol released by 105 cells/90 min. Results were further calculated as a percentage of maximum stimulated rate of lipolysis, 0% representing basal lipolysis.
Materials
Chemicals
Purified human serum albumin was obtained from Hoechst Behring UK Ltd (Hounslow, Middlesex, UK) (free of growth factors including IGF-I and insulin); collagenase from Clostridium histolyticum (batch No C-6885), phloretin, noradrenaline all from Sigma (London, UK). Crystalline porcine insulin and 125I-mono-iodoinsulin with the labelled iodine in tyrosine A14 were both from Novo Nordisk A/S, Bagsvaerd, Denmark. 3-O-methyl-D-[U14C]glucose (specific activity of 126 mCi/mmol) was obtained from Amersham Int. plc (Bucks, UK) and silicone oil 200/50 from Dow Corning Corp. (Poole, Dorset, UK).
Statistics
Statistical analyses were performed using the Student's paired t-test or MannWhitney U-test as appropriate. Linear regression analysis was performed to assess correlation between variables. All results are expressed as mean ± SEM unless otherwise indicated.
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Results |
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Lipolysis inhibition
The maximum percentage lipolysis inhibition observed was 14.92 ± 5.27 in HH and 42.4 ± 8.58 in control subjects and this was significantly different (P < 0.05).
Plasma and metabolite data
The plasma and metabolic data are shown in Table III. Fasting insulin concentrations were higher in HH compared to controls although this did not reach statistical significance (9.9 ± 3.9 mIU/l and 6.2 ± 1.0 mIU/l respectively. There was no significant difference between fasting glucose, NEFA, pyruvate, lactate, alanine, glycerol and hydroxybuytrate concentrations in HH compared to controls. The mean cholesterol concentrations in HH and control subjects were 5.3 ± 0.3 and 4.6 ± 0.2 mmol/l respectively and this approached statistical significance (P = 0.051). The mean triglyceride concentrations were 1.1 ± 0.2 and 0.7 ± 0.1 mmol/l respectively and this was significantly different (P < 0.05) between the two groups. The mean high density lipoprotein (HDL) cholesterol concentration in HH and control subjects was 1.5 ± 0.1 and 1.7 ± 0.2 mmol/l respectively and this was not significantly different between the two groups. The mean low density lipoprotein (LDL) cholesterol concentration in HH and control subjects was 3.4 ± 0.3 and 2.5 ± 0.2 mmol/l respectively and this was significantly different (P < 0.05).
In-vivo insulin sensitivity
The in-vivo resistance index using the HOMA method (Matthews et al., 1985) showed the HH subjects to be modestly but not significantly more insulin resistant than the control subjects (12.29 ± 6.31 versus 4.93 ± 0.92 respectively) (Table III
).
Endocrine parameters
Endocrine data are shown in Table I. The median serum oestradiol concentrations were 70 and 195 pmol/l in patients with HH and control subjects respectively. The progesterone concentrations were all <1 nmol/l in all cases of HH reflecting chronic anovulation. In the control group progesterone concentrations were very low reflecting the early stage of the follicular phase of the menstrual cycle but were slightly higher than the HH group possibly reflecting active ovaries undergoing regular ovulatory cycles and almost certainly a persistent production of progesterone from waning or residual corpus lutea. The median testosterone concentrations in HH and control subjects were 1.2 and 1.3 nmol/l respectively. The mean androstenedione concentrations were 5.5 ± 0.5 and 6.3 ± 0.7 nmol/l respectively and did not differ significantly between the two groups. The expected differences in serum LH and serum FSH were seen between HH and control subjects i.e. LH: 1.5 ± 0.2 and 7.8 ± 2.3 IU/l respectively (P < 0.05), FSH: 1.7 ± 0.3 and 4.0 ± 0.6 IU/l respectively (P = 0.01).
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Discussion |
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Insulin action and ovarian function appear to be intrinsically linked. There is considerable evidence for insulin insensitivity having an effect on ovarian function. Patients with syndromes of severe insulin resistance with acanthosis nigricans, where a genetic cause for the defect in insulin action is established, are usually hirsute and invariably have large polycystic ovaries suggesting that insulin resistance may cause ovarian dysfunction and alter ovarian morphology (Poretsky and Kalin, 1987). Strong evidence for a physiological link between insulin action and ovarian function has come from in-vitro studies. Insulin receptors have been demonstrated on all types of ovarian cells (Poretsky et al., 1985
) and insulin has been shown to augment oestradiol production from granulosa cells and progesterone production from theca cells (Barbieri et al., 1984
; Poretsky and Kalin, 1987
). In-vivo studies have shown patients with PCOS are insulin resistant (Burghen et al., 1980
; Chang et al., 1983
; Dunaif et al., 1989
). A recent in-vitro study of six lean patients with PCOS and eight lean control subjects has confirmed a profound reduction in insulin sensitivity, independent of obesity, with a reduction in insulin receptor binding and a post-receptor defect in insulin action. Maximum adipocyte specific insulin receptor binding was significantly reduced in the patients with PCOS compared to control subjects (0.79 ± 0.17 % for the PCOS group and 1.85 ± 0.14 % for the control subjects per 10 cm2 cell membrane). This difference was highly significant (P < 0.001). Adipocyte insulin stimulated maximum 3-O-methylglucose transport was significantly reduced in subjects with PCOS compared to control subjects [0.86 ± 0.24 and 1.80 ± 0.19 pmol/l per 10 cm2 membrane in PCOS subjects and controls respectively (P = 0.009)]. The ability of insulin to inhibit lipolysis in adipoctyes was impaired in PCOS compared to control subjects [maximum percentage lipolysis inhibition observed was 7.33 ± 2.48% in PCOS and 42.40 ± 8.58% in control subjects (P = 0.006)] (Marsden et al., 1994
). The only two other in-vitro studies investigating insulin resistance in PCOS at the cellular level (Ciaraldi et al., 1992
; Dunaif et al., 1992
) also demonstrated a post-receptor defect in adipocyte insulin sensitivity but did not demonstrate a change in insulin binding. It is noteworthy that both of these studies failed to show a difference in insulin binding between obese and lean normal control subjects or obese and lean PCOS subjects despite large group sizes. All other studies involving examination of the effect of body weight have demonstrated major differences in insulin receptor binding (Olefsky, 1976
; Kolterman et al., 1979
; Wigand and Blackard, 1979
; Pedersen et al., 1981
) and therefore the results of their insulin binding data must be viewed with caution.
Unlike the aforementioned syndromes of insulin resistance the primary aetiology in PCOS remains undefined. The role of anovulation as an aetiological factor has been proposed since observations of women with PCOS have suggested that associated endocrine abnormalities, such as hyperprolactinaemia (Murdoch et al., 1986), raised gonadotrophins and hyperandrogenaemia, improve after ovulation (Baird et al., 1977
; Blankstein et al., 1987
). In-vivo studies of insulin action showed more pronounced abnormalities in amenorrhoeic compared to oligomenorrhoeic women (Kustin et al., 1987
) and more recently in-vivo studies have demonstrated a firm association between reduced insulin sensitivity and anovulatory cycles (Sharp et al., 1991
; Rittmaster et al., 1993
; Robinson et al., 1993
). A recent study from this department using patients as their own controls (studied on two occasions, firstly during amenorrhoea and then again after ovulation) has demonstrated that the marked reduction in insulin sensitivity in PCOS appears to ameliorate after an ovulatory cycle (Marsden et al., 1999
). Therefore insulin resistance and anovulation appear to be intrinsically linked and anovulation has a modifying effect on insulin sensitivity.
Interestingly the in-vitro studies performed on the patients with HH in the studies presented in this paper demonstrate a degree of insulin resistance similar to the in-vitro studies on lean patients with PCOS. It is tempting to speculate that chronic anovulation may have an aetiological role in the insulin resistance associated with PCOS and HH since both these groups of patients are in a chronic anovulatory state. However the situation is far more complex. Firstly, both groups of patients present with amenorrhoea but they have a completely different hormone and biochemical profile. Patients with PCOS are known to have normal or elevated oestradiol concentrations and raised LH concentrations. Steroid hormones per se are known to affect insulin sensitivity. In pregnancy, where oestrogen and progesterone concentrations are markedly raised, carbohydrate, lipid and intermediary metabolism is altered and insulin sensitivity is impaired (Ryan et al., 1985; Stanley et al., 1998
). Artificially raised concentrations of the sex steroid hormones oestrogen and progesterone found in women taking the combined oral contraceptive pill also affect glucose tolerance and produce a degree of insulin resistance (Kasdorf and Kalkhoff, 1988
). In addition, in-vitro studies have recently shown a reduction in insulin receptor binding in the luteal phase of the menstrual cycle (Marsden et al., 1996
) suggesting that progesterone produced in the luteal phase may decrease insulin sensitivity. Patients with HH have very low oestradiol concentrations and both patients with PCOS (anovulatory) and HH have low progesterone concentrations. This suggests that if there is a link between anovulation and insulin resistance then it is unlikely to be simply related to circulating sex steroid concentrations.
Secondly as discussed earlier polycystic ovaries are found in conditions of severe insulin resistance with primary genetic causes for the defect in insulin action. This suggests that insulin resistance per se at least modifies ovarian function. The mechanism by which insulin sensitivity is altered by anovulation is likely to involve factors other than sex steroids.
Androstenedione concentrations did not differ between patients with HH and control subjects. This may be considered surprising since androstenedione is considered to be under the regulatory control of LH which was found to be significantly lower in HH subjects compared to controls in this study. However the production of androstenedione is complex and different control mechanisms exist. Androstenedione is predominantly produced by the ovary and although its control is regulated to a degree by LH, local control through growth factors also plays a significant part. In addition androstenedione is also a derivative of dehydroepiandrosterone (DHEA, produced by the adrenal gland and under the control of adrenocorticotrophic hormone) and the contribution of androstenedione from DHEA in HH is difficult to quantify. Therefore the relationship between LH concentrations and androstenedione concentrations is not clear but the above mechanisms may explain the higher than expected values of androstenedione in these patients.
A significant increase in triglycerides and LDL cholesterol was noted in HH subjects compared to control subjects and although the absolute values in subjects with HH remained in the normal range a trend towards lipid profiles associated with ischaemic heart disease was noted (Bainton et al., 1982). This is interesting since abnormal lipid profiles are seen in other conditions associated with insulin resistance (Bavenholm et al., 1995
). In HH, it remains to be determined to what extent the adverse lipid profile and predisposition to cardiovascular disease is related to hypo-oestrogenaemia and how much to the demonstrated insulin resistance.
In conclusion this study shows that patients with HH appear to have a degree of impaired adipocyte insulin sensitivity similar to that demonstrated in in-vitro studies investigating insulin resistance in amenorrhoeic patients with PCOS. A direct comparison between PCOS and HH was not intended by this paper. However the observations of a reduction in cellular insulin sensitivity in both conditions suggest a consistent direct effect of anovulation on insulin sensitivity.
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Notes |
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References |
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Submitted on January 12, 2000; accepted on May 4, 2000.