Serum Prolactrin and Blood Glucose Levels Before and After an Oral Glucse-load in Patients with Diabetes Mellitus and Liver Cirrhosis

Serum prolactin and blood glucose levels were measured in 13 patients with type II diabetes mellitus and nine patients with liver cirrhosis (Bilharzial or post-hepatic or mixed) in addition to 13 normal controls. Basal serum prolactin levels in diabetic patients did not differ significantly from levels reported for control, while those of liver cirrhosis patients were significantly higher than those of both diabetic patients and controls. After oral glucose load, all the patients showed decreased glucose tolerance accompanied with hyperprolactinemia that was more apparent and persistent in liver-cirrhosis patients. Serum insulin levels that were feasible to be measured in six liver-cirrhosis patients, showed concomitant delayed insulin rise following the glucose load that was maintained longer than in control. These findings of glucose intolerance and hyperprolactinemia were discussed and interpreted on a suggestion to be secondary to various mechanisms including peripheral insulin resistance, gluconeogenesis and/or altered response of pancreatic B-cells to glucose loading.


INTRODUCTION
Pituitary hormone and cytokine Prolactin (PRL) is one of the mediators of the bidirectional communication between neuroendocrine and immune systems.It participates in many immunomodulatory activities, affects differentiation and maturation of both, B and T lymphocytes and enhances inflammatory responses and production of immunoglobulins.Hyperprolactinemia has been described in many autoimmune diseases and the activity of PRL has been intensively studied.Nevertheless, no data on PRL contribution to pathogenesis of diabetes mellitus is available, although the effect of PRL on beta cells of the pancreas and insulin secretion has been observed (Cejkovaet al., 2009).
The effects of (PRL) on lactation and reproductive organs are well known.However, its effects on other target organs and its physiological consequences remain poorly understood.Prolactin was shown earlier to act as a stimulating factor for the immune system and thus it might influence the development of autoimmune diseases, as in type-1 diabetes mellitus.PRL might affect the associated immunological changes occurring in theearly phases of developing type-1 DM (Hala et al., 2010).
Among the various physiological factors known to augment prolactin, insulin induced hypoglycemia which results in significant release of prolactin in normal subjects.This effect is similar to the response of thyrotropine releasing hormone on chlorpromazine, certain dietary factors may either directly or indirectly modify the prolactin response.Prolactin release following hypoglycemia is impaired in presence of massive obesity (Koplemanet al., 1979).
Chronic suppression of prolactin during winter or summer did not significantly alter the amount of insulin released, nor did it significantly alter body composition, so that elevated levels of prolactin in summer do not enhance the release of insulin to glucose (McMahon et al., 1997).Prolactin improves glucose homeostasis by increasing β-cell mass in certain conditions such as pregnancy whereas hyperprolactinemia due to a pituitary gland adenoma tumor exacerbates insulin resistance.
However, previous studies have not evaluated how prolactin modulates β-cell function and insulin sensitivity at different dosages (Landgrafet al., 1975).
The mechanisms by which lactogenic hormones promote β-cell expansion remain poorly understood.Because Prolactin (PRL) up-regulates β-cell glucose transporter 2, glucokinase and pyruvate dehydrogenase activities, Glucose availability might mediate or modulate the effects of PRL on β-cell mass through expression of cell cyclins, cell cycle inhibitors and various other genes known to regulate β-cell replication, including insulin receptor substrate 2, IGF-II, menin, forkhead box protein M1, tryptophan hydroxylase 1 and the PRL receptor (John Wiley and Sons, 2011).Differential effects on gene expression are associated with synergistic effects of glucose and PRL on islet DNA synthesis.PRL up-regulates β-cell glucose uptake and utilization, whereas glucose increases islet PRL receptor expression and potentates the effects of PRL on cell cycle gene expression and DNA synthesis.These novel targets for prevention of neonatal glucose intolerance and gestational diabetes and may provide new insight into the pathogenesis of βcell hyperplasia in obese subjects with insulin resistance (Arumugam et al., 2011).Glucose intolerance and hyperinsulinemia frequently occur in patients with chronic liver failure.Immunoreactive insulin was significantly higher in cirrhotic patients than in controls both before and during the oral glucose tolerance test.As basal C-peptide values were significantly higher and C-peptide/Immunoreactive insulin ratio was significantly lower in cirrhotic patients than in the control subjects.In chronic liver disease investigators failed to establish a pathogenetic role of hormones involved in the glucose counter regulating system.Free fatty acids may play an important role in glucose intolerance in chronic liver failure (Riggio et al., 2004).
The association between prolactin and diabetic retinopathy has been always a matter of controversy.Considerable number of studies performed on this issue and demonstrated increased, decreased, or normal prolactin in patients with diabetic retinopathy (Triebel et al., 2011).Prolactin is the main source vasoinhibins, study of vasoinhibins in animal models demonstrated that these proteins, derived from prolactin hormone could decrease vasopermiability and antagonize the proangiogenic effects of vascularendothelial growth factor and they concluded that diabetic patients had lower levelof this protein (Garcia et al., 2008).
The present study screens the changes in the level of serum prolactin after overnight fasting and following a glucose load dose (tolerance dose) in normal, diabetic patients (who were decided to be treated with hypoglycemic drugs) and liver cirrhosis (Bilharzial or post-hepatic or mixed), also to investigate the implication of prolactin in glucose intolerance.

SUBJECTS AND METHODS
The subjects of the present study included two groups of patients and a control group.The first group included 13 patients (six males and seven females) representing the group of patients suffering from diabetes mellitus.None of the female participants were pregnant or lactating Their ages varied between 40 and 65 years and proved to have type-II DM according to the new WHO criteria for diagnosis of diabetes mellitus and hyperglycemia which is fasting plasma glucose to ≥7.0 mmol/L with symptoms are typical of diabetes, or 2 h post-glucose or casual postprandial plasma glucose level of ≥11.1 mmol/L.If there are no symptoms, or symptoms are equivocal, at least one additional glucose measurement (preferably fasting) on a different day with a value in the diabetic range is necessary to confirm the diagnosis (Keen, 1998).
American Diabetes Association (ADA) issued diagnostic criteria for diabetes mellitus in 1997, with follow-up in 2003 and 2010.The diagnosis is based on one of four abnormalities: hemoglobin A1C (A1C), Fasting Plasma Glucose (FPG), elevated random glucose with symptoms, or abnormal Oral Glucose Tolerance Test (OGTT) (Alberti and Zimmet, 1998).They decided to be treated by oral hypoglycemic drugs.The study conducted from April to December 2011.The second group involved nine males representing the group of patients suffering from liver cirrhosis (Bilharzial or post-hepatic or mixed form) proved by clinical laboratory tests (liver function tests), abdominal ultrasound and liver biopsy.Their ages varied between 35 and 60 years.All patients in our study were normotensive, had normal renal functions with no history of use of drugs that can affect serum prolactin level.All were chosen from the newly administered inpatients of internal medicine department of Assuit University hospital.
The control group comprised of 13 volunteers (nine males and four females) and was more or less matching the patients in age and socioeconomic class.This study is carried out after approval of the research ethics committee of Faculty of Medicine, Assuit University and after taking consent from the participants.
After an overnight fast an oral glucose load dose (1.5 g/kg body weight) were given to all participants (patients and control).At 9.0 a.m.blood samples were taken just before the oral glucose load (0 time, i.e., basal level) and 30 and 90 min after the ingestion of the load dose.Blood glucose was determined by glucose oxidase method using the kits provided by bio Matrix Laboratory reagents and products.For determination of serum prolactin, blood samples were withdrawn without the use of anticoagulant, sera were separated and the concentrations were determined by immunometric assay methods.Catalog LKPR1 Glun Rhonwy, ianberis, Gwynedd, United Kingdom.
It was also possible in this study to follow the pattern of serum insulin concentration in six participants of the control group and another six belongs to the liver cirrhosis group and their data will be included separately in the results.Insulin concentrations were determined by radioimmunoassay method using the human serum insulin kits purchased from Immuno Nuclear Corporation Cat No. 06-l2 Post No. xpo.297, 5 I/USA TER; MI NN ESO TA, USA.

Statistical analysis:
Data obtained in this study were analyzed by statistical analysis using both student ''t'' test and paired ''t'' test.Using SPSS program and p value less than 0.05 is considered significant.

RESULTS
The results of blood glucose (mg/100 mL) in the control group showed significant increase of blood glucose after 30 min ingestion compared to ''0'' min, (p<0.001),Ninety minutes after, blood glucose level showed the tendency to return to the basal levels, where the difference from basal was not significant (Table 1 and Fig. 1a).On the other hand, following glucose load individual values of serum prolactin showed variable response at the 30 and 90 min time (increase, decrease or no change), but the mean value did not differ significantly from the basal (Table 1 and Fig. 1b).There was positive correlation between glucose and prolactin in the basal state (r = 0.44) while after glucose load at 30 min there was negative correlation (r = -0.61)and return positive correlation at 90 min (r = 0.57) (Table 2).No significant difference was found in the level of serum prolactin between males and females (data not showed).
The results of blood glucose (mg/100 mL) in the diabetic group at 0, 30 and 90 min after glucose ingestion revealed significant increase in blood glucose after 30 and 90 min of glucose ingestion (p<0.001 and p<0.01), respectively (Table 3 and Fig. 2a).On the other hand, Prolactin levels significantly increased from the basal at 30 min (p<0.001).After 90 min, the level was decreased significantly from that of 30 min (p<0.01)and although it appeared higher than basal, the difference was insignificant (Table 3 and Fig. 2b).There was negative correlation between glucose and prolactin at '0', at 30 and 90 min after glucose load (r = -0.135,-0.43, -0.12) (Table 2).
The results of blood glucose (mg/100 mL) and serum prolactin (ng/mL) in liver cirrhosis patients at 0, 30 and 90 min after glucose ingestion showed    4 and Fig. 3a).This change was not accompanied with a significant change in serum prolactin level (Table 4 and Fig. 3b).There was positive correlation at '0' state (r = 0.03) meanwhile there were negative correlations afterglucose load at 30 and 90 min r = -0.12 and -0.49, respectively.
The comparison between blood glucose levels in all studied groups showed that basal glucose levels of patients with liver cirrhosis did not differ significantly from those of control.Those of diabetic patients were found to be significantly higher than control (p<0.001) and than those of liver cirrhosis (Table 5 and Fig. 4a).
Statistical comparison between these two groups ofpatients showed that the mean values of diabetic Fig. 5: Prolactin levels (ng/mL) at various periods (glucose tolerance) in the three groups studied (a) fasting prolactin levels (ng/100 mL) in the three studied groups p<0.001 in diabetic and p<0.01 in liver cirrhosis, (b) prolactin levels (ng/mL) 30 after ingestion of load dose in the three studied groups, p<0.01 in diabetic group and p<0.001 in liver cirrhosis group, (c) prolactin levels (ng/mL) 90 min after ingestion of load dose in the three studied groups, p<0.001 in liver cirrhosis patients were significantly higher than those reported for liver cirrhosis patients at both the 30 and 90 min (p<0.001and p<0.001), respectively.In the meantime, the glucose levels of patients (diabetic or cirrhotic) were significantly higher than those reported for control group at 30 and 90 min (p<0.001,0.001 and p<0.001), respectively (Table 5 and Fig. 4b and c).
Comparison between serum prolactin at various periods of glucose tolerance in all groups studied revealed that the basal serum prolactin levels of the diabetic patients although were higher than those of control, they did not differ significantly.Those of livercirrhosis patients were significantly higher than those of control (p<0.001)(Table 6 and Fig. 5a).In diabetic patients, glucose load resulted in a significant rise in serum prolactin at 30 and 90 min comparing to the control group (p<0.001 and p<0.01), respectively.Similarly on comparison of 30 and 90 min serum prolactin levels of liver cirrhosis patients versus those obtained from control revealed that both levels were significantly higher (p<0.001 and p<0.01), respectively (Table 6 and Fig. 5b and c).
Statistical comparison of the serum prolactin levels for the liver cirrhosis patients versus those of diabetes showed that the basal level of Liver cirrhosis group was significantly higher than the diabetic group, (p<0.05)(Table 6 and Fig. 5a), while levels at 30 and 90 min following glucose load did not differ significantly (Table 6 and Fig. 5b and c).
Serum insulin levels determined in six control and six liver cirrhosis patients, the data showed that in the control participants, following glucose load, the mean serum insulin level was markedly raised to a level at 30 min period (p<0.001) then decreased to reach its mean basal level at 90 min (Table 7 and Fig. 6).Therewere negative correlations between insulin, glucose and prolactin in fasting state (r = -0.23 and -0.69), respectively meanwhile there were positive correlations after glucose load at 30 and 90 min (r = 0.61, 0.16, 0.42, 0.41, -0.57, 0.505, 0.50 and 0.31) (Table 8).
In liver cirrhosis patients, the mean fasting serum insulin level appeared to be higher than that of control, but the difference was not significant, following glucose load, there was a delayed insulin response thatmaintained till the 90 min period.The 30 min level appeared to be significantly lower than those of control, (p<0.001) while that at 90 min was significantly higher (p<0.001)(Table 9 and Fig. 6 and 7).There were negative correlations between insulin, glucose and prolactin at '0' state (r = -0.636and -0.44) and return Table 7: Glucose levels (mg/100 mL) and prolactin (ng/mL) and insulin (U/mL) in six males normal subjects Number Blood glucose (mg/100 mL)     8).

DISCUSSION
The effect of raised prolactin levels on glucose homeostasis and insulin release is still not well known.However, its effects on other target organs and its physiological consequences remain poorly understood.Prolactin was shown earlier to act as a stimulating factor for the immune system and thus it might influence the development of autoimmune diseases, as in type-1 diabetes mellitus.Prolactin might affect the associated immunological changes occurring in the early phases of developing type-1 as indicated by the significant reduction of IL-1b and IFN-g (Hala et al., 2010).Shokoofehet al. (2012) demonstrated that all diabetics had higher level of circulatingprolactin than the control subjects and those without retinopathy had higher concentration compared with patients with proliferative retinopathy and those who had severe non proliferative retinopathy had lower prolactin concentration.
Our studies showed that the basal glucose level, serum prolactin and serum insulin in the control group are more or less combatable with the previous studies (Garcia-Webb and Peter Moore, 1999;Charles et al., 1996).After 30 min of glucose ingestion, there is a significant rise in both blood glucose and serum insulin levels (p<0.001) and p<0.001).On the other hand, serum prolactin showed insignificant rise.After 90 min of glucose ingestion, the three parameters of the study return nearly to their basal levels.
In the diabetic group, the basal glucose level showed significant rise (p<0.001)comparing to the control group, whereas serum prolactin showed no significant variation.After 30 min of glucose ingestion, both blood glucose and serum prolactin showed significant rise (p<0.01)respectively comparing to their basal level.At 90 min, the blood glucose level showed significant rise (p<0.01)comparing to its basal level, but serum prolactin showed insignificant rise.It showed that after a glucose load diabetic patients exhibited a markedly reduced glucose tolerance accompanied with hyperprolactinemia.
Patients with liver cirrhosis seem to suffer from hyperglycemia despite the abnormal raised insulin level.Glucose load led to glucose intolerance aided by delayed insulin response that was apparently insufficient to allow proper utilization.The response to glucose load in liver cirrhosis patients was shown by rise in the serum prolactin at 30 min that was variable among individuals, but the mean was not significantly different from the basal level.After 90 min individual levels tended to return to their basal levels or even lower, but the mean serum prolactin was insignificantly lower than the basal level.
The pattern of the 24 h level of plasma prolactin in a group of relatively stable insulin-dependent diabetic patients ''juvenile diabetes' was found by Hanssen et al. (1978) to be similar to that of non diabetes and was unaffected by somatostatin infusion.They also found that plasma prolactin in the normal showed a number of small rises during day and night and that the mean night level was significantly higher than the day time level.
Chronic suppression of prolactin during winter or summer did not significantly alter the amount of insulin released, nor did it significantly alter body composition.Furthermore, acute administration of prolactin did not significantly enhance the release of insulin, during winter or summer treatment periods.Elevated levels of prolactin in summer do not enhance the release of insulin to glucose (McMahon et al., 1997).
In vitro lactogen treatment, in the form of oral prolactin alters insulin secretary behavior and B cell junctional communication and supports our hypothesis that lactogen, insulin secretion and junctional communication among B cells are related (Sorenson et al., 1987).Hyperprolactinemia decreases glucose tolerance via an increase in insulin resistance.
The role of prolactin in the pathogenesis of a human diabetogenic syndrome has been suggested by the studies of Landgrafet al. (1975).These investigators studied the blood glucose and insulin levels during Oral Glucose Tolerance Test (OGTT) glucose and insulin levels were measured in 26 patients with prolactinproducing pituitary tumors.The basal glucose and insulin levels were similar to the control despite chronic hyperprolactinemia.However, GTT was impaired and accompanied by a relative peripheral insensitivity as reflected by the associated hyperinsulinaemia.Even both glucose intolerance and hyperinsulinemia improved after suppression of prolactin release following treatment by bromocriptine.Prolactin injections promoted β-cell mass by increasing β-cell proliferation and neogenesis through the potentiation of phosphorylation of signal transducer and activator of transcription-5 and decreased menin expression.However, only the low-dose prolactin injection potentates glucose-stimulated insulin secretion through glucokinase and glucose transporter-2 induction.In addition, low-dose prolactin decreased hepatic glucose output in hyperinsulinemic states, indicating an improvement in hepatic insulin resistance.However, high-dose prolactin injection exacerbated whole body and hepatic insulin resistance.In contrast to the normal adaptive increases in glucose-stimulated insulin secretion through expanded β-cell mass and insulin sensitivity realized with moderately increased prolactin levels, high levels of prolactin exacerbate insulin resistance and impairs insulin secretary capacity (John Wiley and Sons, 2011).
Glucose intolerance and hyperinsulinemia frequently occur in patients with chronic liver failure.Immunoreactive insulin was significantly higher in cirrhotic patients than in controls both before and during the oral glucose tolerance test.Both hyperproduction and hypo degradation seem to be responsible for the high insulin levels.High basal growth hormone and free fatty acids values were observed in the cirrhotic group.In chronic liver disease free fatty acids may play an important role in glucose intolerance in chronic liver failure (Manabu and Takao, 1996).
Prolactin is known to have renal sodium retention properties in man, since hyperprolactinemia is frequently observed in liver disease, Alain et al. (1994).The mechanisms by which lactogenic hormones promote β-cell expansion remain poorly understood.Because Prolactin (PRL) up-regulates β-cell glucose transporter 2, glucokinase and pyruvate dehydrogenase activities, glucose availability might mediate or modulate the effects of PRL on β-cell mass.

CONCLUSION
It seemed from this study that our patients (diabetic and liver cirrhosis) showed a decrease in glucose tolerance accompanied with hyperinsulinemia and hyperprolactinemia following glucose load.These findings (hyperprolactinemia and hyperinsulinemia) might be explained by increased gluconeogenesis in the liver, enhanced peripheral resistance to insulin and/or altered response of the pancreatic B cells to glucose loading.Prolactin (PRL) up-regulates β-cell glucose transporter 2, glucokinase and pyruvate dehydrogenase activities, glucose availability might mediate or modulate the effects of PRL on β-cell mass.

Fig. 4 :
Fig.4: Glucose tolerance in the three studied groups (a) fasting blood glucose (mg/100 mL) in the three studied groups in group 2: basal value p<0.001,(b) blood glucose values 30 min after ingestion of load dose in the three studied groups p<0.001 in diabetic and liver cirrhosis, (c) blood glucose values 90 min after ingestion of load dose in the three studied groups p<0.001 in diabetic and liver cirrhosis significant rise in blood glucose at 30 and 90 min (p<0.001)and (p<0.001),respectively (Table4and Fig.3a).This change was not accompanied with a significant change in serum prolactin level (Table4and Fig.3b).There was positive correlation at '0' state (r = 0.03) meanwhile there were negative correlations afterglucose load at 30 and 90 min r = -0.12 and -0.49, respectively.

Fig. 6 :
Fig.6: Insulin (uU/mL) in six males normal subjects serum insulin level was markedly raised to a peak level at 30 min period (p<0.001)

Table 1 :
The individual, mean, standard deviation and standard error values of blood glucose (mg/100 mL) and prolactin (ng/mL) obtained from control group

Table 2 :
Correlations between blood glucose (mg/100 mL) and prolactin (ng/mL) in control and diabetic Control

Table 3 :
The individual, mean, standard deviation and standard error values of blood glucose (mg/100 mL) and prolactin (ng/mL) in diabetic patients Fig.1: Mean values of blood glucose (mg/100 mL) and prolactin (ng/mL) in control group (a) blood glucose level after 30 min after ingestion of glucose load p<0.001,(b) prolactin (ng/mL) in control group p value NS

Table 4 :
The individual, mean, standard deviation and standard error values of blood glucose (mg/100 mL) and prolactin (ng/mL) in liver cirrhosis patients

Table 6 :
Prolactin levels (ng/mL) at various periods (glucose tolerance) in the three groups studied

Table 8 :
Correlations between blood glucose (mg/100 mL), prolactin (ng/mL) and insulin (U/mL) in control and liver cirrhosis patients Glucose