DOI: 10.31038/EDMJ.2020442

Abstract

Pituitary abscess (PA) caused by an infectious process is a rare cause of Sellar mass. The clinical features and radiological appearance of PA as an intra- or supra-sellar mass are similar to many other pituitary lesions, and so they are often misdiagnosed as pituitary tumor.

70% of cases occur in a previously healthy pituitary gland. These are classified as primary pituitary abscesses, persumbly secondary to either hematogenous spread or as an extension from an adjacent infective focus such as meningitis, sphenoid sinusitis, Cavernous sinus thrombophlebitis or contaminated cerebrospinal fluid (CSF) fistula.

The rest are secondary abscesses, and arise from pre-existing lesions, such as an adenoma, apoplexy in a tumor, a craniopharyngioma, or a complicated Rathke’s cleft cyst and lymphoma. The risk factors are for PA are immunosuppression, previous irradiation or surgical procedures to the pituitary gland [1].

In almost 50% of cases, the pathogenic microorganism causing the infection is not isolated. A history of recent meningitis sinusitis or head surgery can be the source [2].

Correct diagnosis before surgery is difficult and is usually confirmed intra- or post-operatively. The early surgical intervention allows appropriate antibiotic therapy and hormone replacement resulting in reduced mortality and morbidity. A long term follow-up is recommended because of the high risk of recurrence and of postoperative hormone deficiencies.

Keywords

Pituitary abscess, Papilledema, Panhypopituitarism, Rathke’s cleft cyst, Propionibacterium acnes

Introduction

A pituitary abscess (PA) represents 0.2%-0.6% of all pituitary lesions and can be life threatening. It can have a prolonged disease course. The first case was reported by Heslop in 1848, and so far, <300 cases have been reported worldwide [3]. It is an infectious process that presents as a mass in the Sella. Clinical features and the radiological appearance of the PA as an intra or suprasellar mass are similar to many other pituitary lesions, so it is often misdiagnosed as a cystic pituitary tumor, craniopharyngioma, and Rathke’s cyst. It can be life-threatening if not appropriately diagnosed or treated, and the outcome is difficult to predict. Fortunately, the majority of the cases have a chronic course. The disease has a higher prevalence in females between the age of 12 to 76 years. The average period it takes to diagnose from the onset of symptoms is around 8 years.

PA can occur as a primary disease or can be secondary to infections caused by either hematogenous spread or as an extension from an adjacent infected tissue such as meningitis, sphenoid sinusitis, Cavernous sinus thrombophlebitis or contaminated cerebrospinal fluid fistula 70% of cases occur in a previously healthy pituitary gland. These are classified as primary pituitary abscesses, and the rest are secondary abscesses that arise from pre-existing lesions, such as an adenoma, apoplexy in a tumor, a craniopharyngioma or a complicated Rathke’s cleft cyst and lymphoma [4].

In almost 50% of cases, the pathogenic microorganism causing the infection cannot be isolated. A history of recent meningitis sinusitis or head surgery can be the source [2].

Correct diagnosis before surgery is difficult and is usually confirmed intra- or post-operatively. The early surgical intervention allows appropriate antibiotic therapy and hormone replacement resulting in reduced mortality and morbidity. A long term follow-up is recommended because of the high risk of recurrence and postoperative hormone deficiencies.

We present 2 cases of pituitary abscess in young women. One presented with bilateral papilledema and the other with panhypopituitarism. Both had a sellar mass on an MRI scan, and the diagnosis was made intra-operatively. Microbiological culture in both cases was positive for Propionibacterium acnes (P.acnes). P.acnes is a gram-positive organism, a part of the normal skin microbe. This organism is most commonly isolated from wounds following craniotomies after Staphylococcus aureus and streptococcus epidermidis. Low-grade infections can manifest between 3-36 months.

Case 1

A 14-year old South Asian girl presented with a one-month history of worsening frontal headaches that occurred daily, associated with vomiting, nausea, lethargy, photophobia, and sleep disturbance. Aside from well-controlled asthma, she has been previously healthy. There was no recent travel history or infectious contacts. On examination, she appeared alert and active. She had bilateral papilledema, suggesting raised intracranial pressure (ICP). She was apyrexial and systemically well. Her cerebral magnetic resonance imaging (MRI) scan revealed a soft tissue mass in the pituitary fossa extending up towards the optic chiasm, with mild edema in the optic nerve and tracts. The scan also showed an enlarged pituitary gland and thickened stalk. The findings suggest an inflammatory process like hypophysitis, particularly Langerhans cell histiocytosis (LCH) because of her age. There were no other features of LCH. She had a normal liver US and skeletal survey. She had no symptoms of Diabetes insipidus. Her pituitary hormones were normal, including the stimulated cortisol. Her Prolactin was elevated. Her serum sodium and osmolality were normal. Her ESR was slightly raised, but autoantibodies, serum tumor markers, ACE, and the Quantiferon tuberculosis test were negative. Her IgG4 subclass was normal (Table 1). The formal ophthalmology review did not show evidence of bilateral papilledema. Her symptoms improved with oral analgesics, and steroid treatment was not initiated.

Table 1: Results at initial presentation.

                    Short Synacthen test

                   Baseline tests

A repeat MRI scan 3 months later discussed in a multidisciplinary meeting was reported to suggest Rathke’s cleft cyst abscess/ Pituitary abscess (Figures 1 and 2). She underwent a trans-sphenoidal endoscopic pituitary biopsy for diagnosis. The appearances suggested a Rathke’s left cyst and a pituitary abscess. Immunostaining for ACTH, FSH, LH, growth hormone, TSH and Prolactin, chromogranin, synaptophysin, and collagen IV was consistent with anterior pituitary tissue. Microbiological culture on prolonged incubation was positive for Propionibacterium with no acid-fast bacilli growth. TB culture was also negative. She received a 6-week course of antibiotics, including 2 weeks of intravenous ceftriaxone and oral metronidazole followed by 4 weeks of oral co-amoxiclav. Her headaches and vomiting deteriorated after biopsy with a peak CRP of 218 mg/L, which resolved following medical treatment. Imaging with MRI and baseline pituitary function blood tests has since been repeated following the 6 weeks to assess the management’s effectiveness, which showed normal results. The patient reported the resolution of headaches and able to resume full-time schooling.

Figure 1: MRI at presentation.

Figure 2: MRI 3 months after transphenoidal surgery.

Case 2

29 years old Caucasian fine arts student presented to the emergency department with fever, headaches, profuse sweating, tiredness, and blurring of vision. Her symptoms, particularly headaches, had worsened over the last 12 months. She had noticed polydipsia and polyuria. She also had amenorrhoea for twelve months. She was treated at her local hospital twice in the preceding 3 years with symptoms of headaches, fever, weight loss, and vomiting. She had a lumbar puncture 3 times to rule out a possibility of central nervous system infection. On both occasions, she was discharged home after empirical treatment with antibiotics for suspected meningitis. There was no other past medical history. There was no recent travel history or infectious contacts. She was not on any regular medications.

The initial pituitary MRI and contrast-enhanced MRI scan revealed the absence of the posterior pituitary bright spot and a thickened pituitary stalk with a deviation of infundibulum to the right. There was a homogenous hyperintense area within the pituitary gland with no discernable pituitary tissue. This area was hypointense on T2 (Figures 3-5). The differential diagnosis was apoplexy, hypophysitis or a proteinaceous cystic lesion replacing or compressing the pituitary gland. The optic nerves and the chiasm appeared normal. Her investigations confirmed her to have hypopituitarism with Diabetes insipidus (Table 2). Her lumbar puncture showed no CSF abnormality. Her tumor markers and Quantiferon for tuberculosis were negative. The case was discussed in multidisciplinary meeting (MDT) and with empirical diagnosis of hypophysitis, she was started on prednisolone with the replacement of deficient hormones, including Desmopressin. She showed no improvement in her clinical symptoms. A 3 month interval scan showed an increase in the size of the pituitary gland with further thickening of the stalk and optic chiasm displaced superiorly. After the second discussion in MDT, she had a pituitary biopsy. During surgery, soft yellow-white pus-like material was drained after dural incision. The microscopy showed necrotic material with a little amount of compressed anterior pituitary gland, chronic inflammation, and no evidence of adenoma or granuloma or giant cells was found. No acid-fast bacilli or organisms were seen on gram staining, and the culture for TB was negative. There was scanty growth of Propionibacterium acneformis. Her interval scan 3 months later showed complete resolution of the non-enhancing T1 hypertense pituitary tissue with a further decrease in the size of the pituitary gland. She remains on full hormones replacement. She had an insulin tolerance test that confirmed her growth hormone deficiency, and she is now on growth hormone replacement. She remains on hydrocortisone, Thyroxine, female hormone replacement, and Desmopressin.

Figure 3: MRI at presentation.

Figure 4: MRI 3 months later.

Figure 5: MRI post-surgery.

Table 2: Results at initial presentation.

                   Short Synacthen test

                   Baseline tests

Discussion

A pituitary abscess is an infectious process characterized by the accumulation of purulent material in the sella turcica. It is rare, and can be a life-threatening condition unless promptly diagnosed and treated. We report 2 cases of secondary pituitary abscess in young women. The first case was due to abscess in the Rahtke’s cleft cyst (RCC), and the second was Pituitary gland abscess with a history of otitis media and repeated lumbar punctures for presumed meningitis.

The clinical presentation of PA is nonspecific, such as headaches, pituitary hypofunction, and visual disturbances, whereas the infection can be discreet and inconstant [5,6]. Symptoms can be acute, subacute, or chronic, explaining the late diagnosis; in some cases. Visual disturbance, including hemianopia, can be present in 50% of cases. Headache without a particular pattern is a regular feature (70-90%) and can be debilitating. Anterior pituitary hypofunction due to destruction and necrosis of the gland is the commonest presentation resulting in fatigue and amenorrhoea (54-85%). In one series, 28 out of the 33 patients had anterior pituitary hypofunction. Pituitary hormone deficiencies persist in the majority of patients following treatment Up to 70% of patients with PA can have central Diabetes insipidus. In contrast, fever with signs of meningeal irritation is reported in 25% of cases [5].

MRI is the imaging of choice for the pituitary lesions. PA can present as a suprasellar mass (65%) or as an intrasellar mass (35%). A typical PA appears as a single cystic or partially cystic mass that is hypointense on T1-weighted image and hyperintense in T2-weighted image. It can show a rim of enhancement after contrast gadolinium. The posterior pituitary bright spot is mostly absent in majority of the cases (Wang et al.). The lesion’s signal depends on protein, water, lipid content, and whether there is hemorrhage. Imaging can also show the invasion of an adjacent anatomical structure, peripheral meningeal enhancement, thickening of the pituitary stalk, and paranasal sinus enhancement [6].

Diffusion-weighted magnetic resonance imaging (DWI) is widely used to differentiate cerebral abscess from other necrotic masses. Brain abscesses typically show high intensity on DWI with decreased apparent diffusion coefficient (ADC) value in their central region. The high intensity on DWI is useful but not specific to PA because pituitary apoplexy can also exhibit high intensity on DWI [7]. The accuracy of DWI in PA remains controversial. In the Wang et al. case series, PA was misdiagnosed in one-third of the case [6]. The radiological differential diagnosis includes, Rathke’s cleft cyst, cystic pituitary adenoma, arachnoid, and dermoid cysts, metastases, glioblastoma multiforme, chronic hematoma, and multiple sclerosis [8]. Rathke’s cleft cyst mainly can mimic a pituitary abscess [9]. RCC is the second most common incidentaloma after adenomas and accounts for 20% of incidental pituitary lesions at autopsy. The incidence of RCCs in children was reported to be much lower than in adults. However, the prevalence is now believed to be much higher, especially among those with the endocrine-related disorder [10]. Gunes et al. reported the radiological appearance of RCC on MRI in 13.5% of the children who underwent MRI for the investigation of endocrine-related disorders. Patients with RCC are usually asymptomatic, but symptomatic RCC is more common in females in both adult and pediatric populations [11]. RCC can cause significant morbidity such as headache, visual disturbances, chemical meningitis, endocrine dysfunction (hypothyroidism, menstrual abnormalities, diabetes insipidus, adrenal dysfunction, and very rarely apoplexy). Short stature, growth deceleration, delayed puberty are also reported in children and adolescents.

The diagnosis of PA in most cases can only be confirmed after surgical exploration, due to overlapping of clinical signs, symptoms, imaging, and laboratory findings with other sellar lesions. Signs of inflammation are present in less than a third of the patients. The PA should be included in the differential diagnosis of patients with headaches or signs of pituitary dysfunction and patients with pituitary mass who develop signs of meningeal inflammation.

The main treatment for PA in patients with mass effect is Transsphenoidal excision (TSS) with decompression of sella and antibiotic therapy. This can result in the resolution of visual abnormalities. Treatment is effective for typical symptoms such as fever, headache, and visual changes. Patients with shorter duration of symptoms and those with primary abscess have better improvement in their pituitary dysfunction. Majority of the patients remain with pituitary dysfunction even after the treatment.

Antibiotic therapy should to started promptly even in the patients who are waiting for microbiology and histological confirmation for about 4–6 weeks [1,12]. Empirical treatment with ceftriaxone is indicated until the results are available. Hormone replacement is commenced depending on the hormone deficits including stress dose glucocorticoid therapy. Hypocortisolemia should be recognized among patients presenting with sellar masses, as early diagnosis and treatment improve survival and endocrinological outcome. Patients who suffer from the pituitary abscess may eventually have a good quality of life if they are diagnosed and treated early. A craniotomy is reserved for larger lesions with the suprasellar extension or where transsphenoidal surgery is ineffective [13]. In a series published with 66 patients, 81.8% of patients recovered completely, 12.1% of patients had at least one operation for recurrence, and only one patient had died [14].

There are widespread pathogenic microorganisms in abscesses. These include Gram-positive bacteria, Gram-negative bacteria, anaerobes, and fungi [8,11]. Streptococcus and Staphylococcus are the most predominant Grampositive bacteria, whereas Escherichia coli, Mycobacterium, and Neisseria have also been reported [3,10,11]. Aspergillus fumigatus is mostly isolated in cases of secondary PA. Immunosuppressed patients mostly have Candida and Histoplasma. Cultures are positive only in 50% of cases; therefore, broad-spectrum antibiotics are given as empirical treatment. The pathogen identification is important for the therapeutic management [15].

Both patients had culture-positive for Propionibacterium acnes (P. acnes). This organism is seated deeply in the pilosebaceous glands, mainly in the scalp and face. It is a slow-growing, pleomorphic, non-spore-forming gram-positive anaerobic bacillus that is a universal component of the normal skin microbiota. It is usually considered a contaminant of blood cultures but occasionally can cause serious infections, including postoperative central nervous system (CNS) infections. P. acnes are the most commonly isolated organism after Staphylococcus aureus and Staphylococcus epidermidis following craniotomies. In the presence of heavy infiltrates, the Gram stain is not reliable. Gram stain is only positive in about 10.5% of clinically significant infections with moderate growth. P. acnes behave in a less aggressive manner than other postsurgical organisms and only accounts for a small fraction of CNS infections [16]. P. acnes abscesses typically follow craniotomy, shunts, access to reservoirs, trauma, and foreign bodies. Granulomatous responses have been documented in the CNS following P. acnes infections.

P. acnes grow slowly in the laboratory. This can cause in a delay in diagnosis, missed diagnosis, or delay in treatment if specimens are not cultured for an extended period. Cultures may not grow for as long as 14 days, so samples should be held beyond the usual 5 to 7 days. Gram stain may not be a reliable technique for the rapid diagnosis of P. acnes infections. When there is evidence of an abundant inflammatory response in the Gram-stained smear, a more careful evaluation of cultures must be performed. Polymerase chain reaction for the 16S rRNA or mass spectrometry can be a useful tool for rapid identification and typing of P. acnes following recovery in culture. Propionibacterium is susceptible to antibiotics used for the treatment of anaerobic infections, including penicillin, erythromycin, lincomycin, and clindamycin, but not metronidazole, which is notably ineffective against P. acnes [17].

Patients with PA should be followed up with serial MRI of the pituitary, hormonal profile and visual fields at 3, 6, and 12 months after surgery. The recurrence rate is variable and depends on the nature of the abscess (primary or secondary. The majority of relapses are associated with either an immunological defect or previous pituitary surgery [12,18].

Conclusion

We presented 2 cases of unusual sellar mass from an abscess in an adolescent and a young adult due to P. acnes, both responded well to treatment.

The pituitary abscess should be included as the differential diagnosis of patients with a sellar or a suprasellar mass, headaches, pituitary dysfunction, and meningeal inflammation.

The diagnosis is difficult before surgery because of overlapping clinical signs, radiological and laboratory findings with other sellar lesions.

Broad-spectrum antibiotics should be started empirically even before the culture results are available.

Culture is positive only in 50% of cases, and in case of unusual bacteria like P. acnes, an extended culture is required for the confirmation of the diagnosis.

Pituitary dysfunction should be recognized and appropriately treated particularly glucocorticoid replacement.

Transsphenoidal surgery is the treatment of choice and this is followed by pronged 4-6 weeks of broad-spectrum antibiotic therapy.

Early and efficient surgical and medical management results in lower mortality and higher recovery of pituitary hormone function.

Patients should be followed up with MRI imaging, assessment of the hormone replacement if required, and visual field assessment because of a chance of recurrence.

References

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DOI: 10.31038/EDMJ.2020435

Abstract

Aims: Circulating neurotoxic autoantibodies to the 5-hydroxytryptamine 2A receptor were increased in older adult type 2 diabetes in association with certain neurodegenerative complications. The male Zucker diabetic fatty (ZDF) rat is a model system for studies of obese, type 2 diabetes mellitus. The aim of the current study was to test for (and compare) circulating neurotoxic autoantibodies to the 5-hydroxytryptamine 2A receptor in the Zucker diabetic fatty rat and age-matched lean Zucker rat strains.

Methods: Plasma from lean and Zucker diabetic fatty rat (obtained at different developmental stages) was subjected to protein G affinity chromatography. The resulting immunoglobulin G fraction was tested for neurotoxicity (acute neurite retraction, accelerated neuron loss) in N2A mouse neuroblastoma cells and for binding to a linear synthetic peptide corresponding to the second extracellular loop of the 5-hydroxytryptamine 2A receptor.

Results: The male Zucker diabetic fatty rat (fa/fa) and two Zucker lean strains (+/?) and (fa/+) harbored autoantibodies to the 5-hydroxytryptamine 2A receptor which appeared spontaneously around 7-8.5 weeks of age. The circulating autoantibodies persisted until at least 25 weeks of age in the Zucker diabetic fatty rat and in the Zucker heterozygote (fa/+), but were no longer detectable in 25-week-old lean (+/?) Zucker rats. Autoantibody-induced acute neurite retraction and accelerated loss in mouse neuroblastoma N2A cells was dose-dependently prevented by selective antagonists of the 5-hydroxytryptamine 2A receptor. It was also substantially prevented by co-incubation with antagonists of RhoA/Rho kinase-mediated signaling (Y27632) or Gq11/phospholipase C/inositol triphosphate receptor-coupled signaling.

Conclusions: These data suggest that neurotoxic 5-hydroxytryptamine 2A receptor-targeting autoantibodies increase in the aging male Zucker diabetic fatty rat and in male Zucker lean rats harboring a heterozygous mutation, but not in age-matched, older Zucker lean rats lacking a known leptin receptor mutation. The Zucker genetic strain may be useful in studies of the role of humoral and/or innate immunity in late neurodegeneration.

Introduction

Type 2 diabetes mellitus is associated with an increased risk of late neurodegeneration [1,2] via mechanisms which may involve (in part) increased peripheral and central inflammation. Increased pro-inflammatory cytokines and innate immunity have been associated with early Parkinson’s disease [3] and dementia [4] through complex mechanisms. Leptin is a hormone released by fat cells that is important in energy metabolism. Dysfunction of the leptin system results in classic signs of type 2 diabetes mellitus, such as obesity, insulin resistance and high circulating insulin. Leptin is also a member of the type 1 cytokine family that includes interleukin-6 [5,6]. The leptin receptor is normally expressed on hematopoietic cells where it stimulates helper- T cell, and effector T cell functions and inhibits regulatory T cell function [7].

The male Zucker Diabetic Fatty rat (fa/fa) (ZDF) is a well-recognized animal model of obese type 2 diabetes mellitus and hypertension [8,9] in which hyperphagia causes morbid obesity leading to high level of pro-inflammatory adipocytokines [10]. In the ZDF rat, pro-inflammatory cytokines cause enhanced innate immunity [11], however, owing to its complete leptin receptor deficiency, helper T-cell function is reduced [12]. The lean heterozygous Zucker rat (fa/+) has haplo-sufficiency at the leptin receptor and a two-fold increased plasma leptin concentration [13] compared to the wild-type (+/+) lean Zucker rat. Since hyperleptinemia promoted helper-T cell-mediated autoantibody formation and worsened clinical outcome(s) in a mouse model of systemic lupus erythematosus [14], the lean heterozygote Zucker rat might be expected to exhibit higher autoantibody expression than in the ZDF (fa/fa) rat strain.

In prior studies in older adult (human) obese type 2 diabetes, patients harboring increased circulating autoantibodies to the 5-hydroxytryptamine 2A receptor (5-HT2AR) had an increased prevalence of co-morbid neurodegenerative complications including Parkinson’s disease and dementia [15]. Human plasma autoantibodies targeting the 5-HT2A receptor promoted long-lasting (agonistic) activation of Gq11/phospholipase C/inositol triphosphate receptor/Ca2+ signaling causing accelerated endothelial cell and neuronal cell death [16]. Since the 5-hydroxytryptamine 2A receptor is normally expressed on arterial smooth muscle cells [17] and on neurons in specific brain regions involved in the regulation of mood, thinking, perception, and sleep [18], altered signaling at the 5-HT2A receptor could have diverse peripheral and central effects.

The aim of the present study was to test for spontaneously-occurring autoantibodies to the 5-HT2A receptor in the circulation in male ZDF rat (fa/fa) and age-matched male lean Zucker rats either lacking a known leptin receptor mutation (+/?) or harboring a heterozygous leptin receptor mutation (fa/+). The developmental expression of autoantibody was compared in each genetic strain, and the mechanism of neurotoxicity downstream of 5-HT2AR binding was evaluated in mouse neuroblastoma N2A cells.

Materials and Methods

Animals

All procedures were conducted according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the Veterans Affairs Medical Center (East Orange, New Jersey). Male ZDF (N=12) and lean (+/?) Zucker rats (N=12) were obtained from Charles River Laboratories (Kingston, NY) at approximately 6 weeks of age. All rats were single housed upon arrival, with modest enrichment (a PVC tube). Rats were provided ad libitum access to food and water and maintained in a 12 h light/dark cycle with lights on at 0630. All procedures occurred during the light phase of the cycle.

Blood Drawing

EDTA plasma from 10-week ZDF rat and 10-week lean, heterozygous (fa/+) Zucker rats was obtained from Charles River Laboratories. For all other time points, blood was collected from the retro-orbital plexus of ZDF, and lean (+/?) Zucker rats using a retro-orbital blood procedure. The blood was centrifuged at 1400 g x 10 minutes at 4 degrees C. The resulting plasma was stored at -4^oC for up to 2-3 weeks prior to protein G affinity chromatography.

Tail-Nick

Capillary blood glucose was determined from a drop of blood obtained after tail nick at sequential time intervals. A glucose dehydrogenase method (Accucheck Aviva, Roche Diagnostics, Inc) was used for the determination of blood glucose.

Protein G Affinity Chromatography

Protein-G affinity chromatography was carried out as previously reported for protein-A affinity chromatography in human plasma [16]. The pH of the 10 mm Tris binding buffer (6.5) and the 0.1 M citrate elution buffer (2.7) was adjusted to optimize binding and elution of rat immunoglobulin G. Protein-G eluate fractions were stored at 0-4 degrees C.

Mouse N2A Neuroblastoma Cells

Cells were cultured in DMEM with 10% fetal calf serum as previously reported [16].

Neurite Retraction and N2A Cell Survival Assays

Assays were performed as previously described [16].

Enzyme-Linked Immunoassay (ELISA)

The ELISA procedure was carried out as previously reported using an 18-meric linear synthetic peptide Q..N18 corresponding to the second extracellular loop of the human 5-HT2AR as the solid phase antigen [15].

Chemicals

All chemicals were obtained from Sigma Co., Inc. (St. Louis, MO).

Protein determination-Protein levels were determined using a bichinchonic assay method (BioRad, Inc.).

Statistics

Statistical analysis was performed using an unpaired Student’s t-test with an α cutoff level for significance of < 0.05.

Results

Occurrence of Circulating 5-HT2A Receptor Autoantibody in the ZDF Rat: Relation to Obesity and Diabetes

Mean body mass in ZDF rats significantly exceeded that in lean (+/?) Zucker rat at week 7, week 8.5, and week 12 (Figure 1A). There was no longer a significant weight difference between the strains at week 25 (Figure 1A). Diabetes (hyperglycemia) emerged between week 8-10 in the ZDF rat, but at week 7 the ZDF rat was still normoglycemic (Figure 1B). In contrast, lean (+/?) Zucker rats were normoglycemic at all ages tested (Figure 1B), not tested at week 7.

Figure 1: Age-dependent change in body mass(A) and capillary blood glucose concentration (B) in male Zucker fatty (ZDF) vs lean (+/?) (ZDL) rats. A) Body weight increased significantly in the male ZDF rat (starting at week 7) compared to control lean Zucker rat B) capillary blood glucose was determined by tail nick using glucose dehydrogenase method as described in Materials and Methods. Glucose was normal at week 7 in the ZDF rat, but increased significantly compared to ZDL rat starting at week 8.

Detection of plasma immunoreactive 5-HT2AR autoantibody had a similar trajectory to diabetes in ZDF rats. At 7 weeks of age, ZDF rat had no detectable plasma immunoreactive 5-HT2AR autoantibody (Figure 1C). At 10-weeks of age, plasma from ZDF rat contained a two-fold, higher binding to 5-HT2AR (0.14 AU, N=2) compared to background levels (0.07 AU; Figure 2a). Plasma from lean ((+/?) Zucker rats harbored two-fold or slightly higher level of autoantibody binding to 5-HT2AR between 8.5-15 weeks of age which declined to undetectable levels at 25 weeks of age (Figure 2B).

Figure 2: Developmental expression of autoantibody binding to 5-HT2AR synthetic peptide in three Zucker rat strains. A 2 ug/mL concentration of the protein G eluate was tested for binding to the linear synthetic 18-meric 5-HT2A receptor peptide as described in Materials and Methods. Results are “mean +/- SE” in A) ZDF rat: 7-week (N=2); 8.5-week (N=3); 10-week (N=2); 25-week (N=3) B) lean (+/?) Zucker rat: 7-week (N=3); 8.5 week (N=3); 15-week (N=10); 25-week (N=6). C) lean heterozygote (fa/+) Zucker rat: 7-week (N=2); 10-week (N=5); 25-week (N=2).

Spontaneous IgG autoantibody to the 5-HT2A receptor was already present at 7 weeks of age in the heterozygous lean (fa/+) Zucker rat (Figure 2C) and levels at 10-weeks old and 25-weeks old were substantially higher than in age-matched ZDF rat (Figure2C). Autoantibody to the 5-HT2A receptor persisted into older age, i.e. 25 weeks old, in male ZDF and lean heterozygous Zucker rats, but disappeared at 25 weeks of age in the lean (+/?) Zucker rat lacking a known leptin receptor mutation. In a recent report [19], diabetes, obesity and hypertension were associated with substantially increased hazard rates for the occurrence of dementia, Parkinson’s disease and severe depression following traumatic brain injury in older adult veterans.

The present data suggest that male ZDF and heterozygous lean rat strains may be useful in modeling the effects of heightened innate and/or adaptive immunity, respectively, on the occurrence of neurodegeneration.

Zucker Diabetic Fatty Rat Plasma 5-HT2AR Autoantibody Inhibits N2A Cell Survival

We next tested for neurotoxicity in the autoantibody (from 10- week-old ZDF rat plasma) that displayed increased binding to the 5-HT2A receptor peptide. Autoantibody in plasma from the 10-week old ZDF rat caused dose-dependent inhibition of survival in N2A mouse neuroblastoma cells (Figure 3). A two and one-half microgram per milliliter concentration of the IgG caused approximately 40% neuroblastoma cell loss (after 24 hours incubation) compared to control, untreated cells (P< 0.01, Figure 3).

Figure 3: Dose-dependent neurotoxicity in the protein G eluate fraction from Zucker diabetes fatty rat plasma. The protein G-eluate fraction from 10-week-old male Zucker diabetic fatty rat plasma was incubated with N2a mouse neuroblastoma cells (at the indicated concentrations) for 24 hours. % N2a cell survival was determined with an MTT assay as described in Materials and Methods. Each point represents the mean + SE of quadruplicate determinations; * P < 0.01 compared to cell survival in untreated cells.

Zucker Diabetic Fatty Rat Plasma 5-HT2AR Autoantibody Causes Acute N2A Neurite Retraction

A 120 nanomolar concentration (1.8 g/mL IgG) of the protein G- eluate fraction of plasma from 10-week-old male ZDF fatty rat caused significant 37% acute neurite length-shortening (after 15 minutes of incubation) of N2A mouse neuroblastoma cells (Figure 4A). Co-incubation with a 200 nanomolar concentration of M100907, a highly selective, potent antagonist of the 5-HT2AR, afforded 86% significant (P < 0.01) protection against autoantibody-induced neurite retraction (Figure 4A).

Figure 4: Neurite outgrowth inhibition (A) and accelerated N2A neuroblastoma cell loss (B) in autoantibody from ZDF rat plasma: neutralization by M100907, a highly selective 5-HT2AR antagonist. A) A 120 nM concentration of the protein-G eluate fraction of 10-week-old male ZDF rat plasma was incubated with N2A mouse neuroblastoma cells in the presence or absence of a 200 nM concentration of the selective 5-HT2AR antagonist M100907. % Basal neurite length was determined after 15 minutes as described in Materials and Methods B) A 160 nM concentration of the protein G-eluate fraction from 10-week-old male Zucker diabetic fatty rat plasma was incubated with N2a mouse neuroblastoma cells in the presence or absence of the indicated concentration of M100907 for 24 hours at 37 deg C. Percent N2A cell survival was determined with an MTT assay as described in Materials and Methods. Each point represents the mean + SE of triplicate determinations.

Pharmacologic Profile of Zucker Diabetic Fatty Rat Plasma 5-HT2AR Autoantibody Toxicity

A ~170 nanomolar concentration (2.5 µg/mL) of the protein G-eluate fraction of plasma from 10-week-old male ZDF rat was incubated with N2A mouse neuroblastoma cells in the presence or absence of a (500 or 1000) nanomolar concentration of M100907 for 24 hours at 37 deg C. M100907 caused significant (dose-dependent) protection against accelerated N2A cell loss, after 24 hours incubation with the ZDF rat IgG (Figure 4B).

Spiperone is a potent, but less selective 5-HT2AR antagonist. Spiperone at 500-1000 nanomolar concentrations afforded significant dose-dependent protection against ZDF rat IgG-induced accelerated N2A cell loss (Figure 5). Ketanserin is a less potent 5-HT2AR antagonist compared to M100907 or spiperone. Substantially higher concentration (2.5 µM) of ketanserin was required to significantly protect against ZDF rat IgG-induced N2A cell loss (Figure 6).

Figure 5: Potent, less selective 5-HT2AR antagonist (spiperone) dose-dependently prevented accelerated neuron loss induced by ZDF rat plasma autoantibody. A 160 nM concentration of the protein G-eluate fraction from 10-week-old male Zucker diabetic fatty rat plasma was incubated with N2a mouse neuroblastoma cells in the presence or absence of the indicated concentration of spiperone for 24 hours at 37 deg C. Percent N2a cell survival was determined with an MTT assay as described in Materials and Methods. Each point represents the mean + SE of triplicate determinations.

Figure 6: Higher concentration of ketanserin, a less potent, but selective 5-HT2AR antagonist dose-dependently prevented accelerated neuron loss induced by ZDF rat plasma autoantibody A-B) 120 nM concentration of the protein G-eluate fraction from 10-week-old male Zucker diabetic fatty rat plasma was incubated with N2a mouse neuroblastoma cells in the presence or absence of the indicated concentration of ketanserin for 24 hours at 37 deg C. Percent N2a cell survival was determined with an MTT assay as described in Materials and Methods. Each point represents the mean + SE of triplicate determinations.

The receptor antagonist profile and concentration associated with significant protection against ZDF IgG autoantibody-induced neuroblastoma cell loss is consistent with relative affinity constants on the 5-HT2A receptor (M100907 < spiperone<< ketanserin).

A one micromolar concentrations of SB-20471, a selective antagonist to the 5-HT2B receptor did not significantly prevent ZDF autoantibody-induced neurite retraction (Table 1). A ten micromolar concentration of either bosentan (an endothelin-1 receptor) or losartan (an angiotensin II type 1 receptor) antagonist did not significantly protect against ZDF IgG-induced acute N2A neurite retraction (Table 1). An 850 nanomolar concentration of prazosin (alpha 1 adrenergic R antagonist) did not afford significant protection against ZDF IgG autoantibody-induced acute neurite retraction (Table 1). Taken together, these data suggest that neurotoxicity in the 10-week old male ZDF rat IgG appears selective for the 5-HT2A receptor.

Table 1: Receptor specificity of prevention of ZDF rat IgG autoantibody-induced acute N2A neurite retraction.

A 160 nanomolar concentration of 10-week-old male Zucker fatty rat IgG autoantibodies was incubated in the presence or absence of the indicated GPCRs. Acute N2A neurite retraction was determined after 15 minutes as described in Materials and Methods. Results are mean +/- SD.

Involvement of Gq11/Phospholipase C/Inositol Triphosphate and Rho Kinase Signaling Pathways

Co-incubation of N2A cells with ZDF plasma IgG autoantibodies together with individual selective antagonists of Gq11 (YM-254890), phospholipase C (U73122), inositol triphosphate receptor (2-APB) signaling or Rho A/Rho kinase (Y27632) mediated signaling completely or nearly completely protected against acute neurite retraction (Table 2) consistent with involvement of Gq11/phospholipase C/inositol triphosphate and Rho kinase signaling pathways in mediating IgG neurotoxicity downstream of 5-HT2A receptor binding.

Table 2: Effect of signaling pathway inhibitors on ZDF rat IgG autoantibody-induced acute N2A neurite retraction.

A 160 nanomolar concentration of 10-week-old male Zucker fatty rat IgG autoantibodies was incubated in the presence or absence of the indicated GPCRs. Acute N2A neurite retraction was determined after 15 minutes as described in Materials and Methods. Results are mean +/- SD as described in Materials and Methods.

Level and Titer of Autoantibody to 5-HT2A Receptor in Male Zucker Heterozygote (fa/+)

An identical (1.5 µg/mL concentration) of the IgG from Zucker heterozygote rat plasma (N=5) displayed two-fold significantly higher binding (to the 5-HT2A receptor peptide) compared to IgG in plasma from five ZDF rat plasmas (N=2, 10-week-old and N=3, 25-week-old rats) (Figure 7A). The titer of autoantibody to the 5-HT2A receptor peptide was more than two-fold higher in the 10-week old lean Zucker heterozygous rat compared to age-matched, 10-week old ZDF rat (Figure 7B).

Figure 7: A) 2 ug/mL concentration of the protein G eluate of ZDF rat and lean Zucker heterozygote plasma was tested for binding to linear, synthetic 18-meric 5-HT2A receptor peptide.
B) Titer of autoantibody binding to 5-HT2A receptor peptide was tested at the indicated concentrations in 10-week lean Zucker heterozygote (N=5) or 10-week ZDF (N=2) protein G eluate. Results are “mean +/- SE”. * P< 0.01

Bioactivity in the Antibody Fraction of Male Zucker Heterozygous (fa/+) Plasma

Dose-dilution curves of the protein-G eluate fraction from three, 10-week-old lean heterozygous Zucker heterozygous plasmas demonstrated potent inhibition of N2A cell survival at all IgG concentrations tested in two of three rats (Figure 8A). In one of the three rats, lean heterozygous Zucker rat #3, a component of significant N2A survival-promoting activity were observed at high IgG dilution (Figure 8A). ‘Functional selectivity’ is a unique property of the 5-hydroxytryptamine 2A receptor [20]-it refers to the ability of different 5-HT2AR ligands to preferentially activate alternative signaling pathways, e.g. PLC/IP3/Ca2+ signaling or a beta-arrestin 2/PI3-kinase survival pathway. Since the selective 5-HT2AR antagonist M100907 significantly protected against lean heterozygous Zucker rat IgG autoantibody-induced accelerated N2A cell loss (Figure 8B) the neurotoxicity is likely due to Gq11/PLC/IP3/Ca2+-mediated signaling occurring downstream of 5-HT2AR binding. More study is needed, however, to determine whether N2A survival-promoting activity in a subset of lean heterozygous Zucker rat IgG autoantibodies may result from selective activation of beta arrestin-2/PI3-kinase-mediated signaling. In a prior report [21], highest level of 5-HT2A receptor-binding occurring in the autoantibodies from a patient with lupus. The lupus IgG autoantibodies were not only neurotoxic, but (at certain concentrations) promoted N2A neuroblastoma cell survival dependent on PI3 kinase-mediated signaling [21].

Figure 8: A) Dose-dilution curves of three Zucker lean heterozygous protein G eluates on survival in N2A mouse neuroblastoma cells; B) Co-incubation of a representative lean heterozygous protein G eluate fraction with 1 µM concentration of the selective 5-HT2AR antagonist M100907. * P< 0.05.

Apparent MW of the Active Component in ZDF Protein G Eluates

The active component in the ZDF protein G eluate (which caused accelerated N2A cell loss) had an apparent MW > 10 kD (Figure 9A) consistent with an antibody or antibody fragment. There was no significant N2A cell survival-inhibitory activity present in the flow-through fraction following membrane dialysis of the ZDF protein G eluate on a 10 kilodalton Molecular Weight (MW) cutoff membrane (Figure 9A). The freshly-isolated, lean heterozygous Zucker rat (fa/+) protein G eluate fraction (SM, starting material) caused significantly increased N2A cell survival. Following dialysis on a 10 kD MW cutoff membrane, more than 90% of the N2A survival stimulatory activity was recovered in the fraction having an apparent MW > 10 kD, i.e. retained on the membrane (Figure 9B). The low MW (< 10 kD) flow-through fractions following membrane dialysis of the ZDF or lean heterozygous Zucker rat protein G eluate did not cause significant inhibition or stimulation of N2A survival (Figure 9A and 9B).

Figure 9: Apparent MW of peak neuroblastoma survival-inhibitory (A) and – stimulatory (B) activity in the plasma protein G eluate from ZDF or heterozygous lean ZDL rat. Apparent MW of peak neuroblastoma survival-inhibitory (A) or -stimulatory (B) activity in the protein G eluate from a representative ZDF (A) and lean heterozygote # 3 Zucker rat (B) plasma. A 160 nM concentration of the protein G-eluate fraction from 10-week-old male Zucker rat plasma was dialyzed on a 10kD MW cutoff membrane. The resulting retained and flow-through fractions or starting material (SM) were incubated with N2a mouse neuroblastoma cells for 24 hours as described in Materials and Methods. Each point represents the mean + SE of triplicate determinations. * P < 0.05 compared to control N2A cell without added test fractions.

Discussion

The male ZDF rat is a well-studied model of human obese type 2 diabetes mellitus. The animals undergo progressive gain in fat mass, with obesity arising around 4-weeks of age [9]. Hyperglycemia commences around week 8- 9, and chronic marked elevation in blood glucose concentrations (400-500 mg/dL) is associated with early hyperinsulinemia followed by later relative insulin deficiency [10]. Hypertension and severe hypertriglyceridemia manifest around week 9 in the ZDF rat [9,10]. In adult, long-standing human obese type 2 diabetes mellitus from the Veterans Affairs Diabetes Trial, the baseline presence of anti-endothelial cell autoantibodies was significantly associated with lack of baseline insulin therapy (i.e. relative insulin deficiency) and lack of baseline triglyceride-lowering medication [22]. Endothelial cell heparanase expression is upregulated in insulin deficiency [23] and by high level of pro-inflammatory cytokines [24]. Heparanase cleaves heparan sulfate side chains abundant on neurons and vascular cells, and heparan sulfate proteoglycan is a known target of humoral autoimmunity [25]. In older adult diabetes patients evidence suggested that autoantibodies targeting the 5-HT2AR were in many cases cross-reactive with heparan sulfate proteoglycan [26]. Taken together it is possible that certain metabolic derangements in the ZDF rat strain, (insulin deficiency, and high level of pro-inflammatory cytokines) may contribute in part to the long persistence of circulating 5-HT2A receptor autoantibodies at older ages.

Although the heterozygous (fa/+) Zucker rat is lean and non-diabetic, it demonstrated substantially higher titer of 5-HT2A receptor autoantibodies compared to age-matched ZDF rat. One possibility is that enhanced helper T-cell immunity in the lean heterozygote Zucker rat drives autoantibody formation which includes a subset (of autoantibodies) targeting the 5-HT2A receptor. Neurotoxicity in the lean heterozygote Zucker IgG autoantibodies (i.e. acute neurite retraction and accelerated N2A cell loss) was as high or higher than in ZDF IgG autoantibodies consistent with a prior report of a significant correlation between antibody binding level and level of acute neurite retraction in N2A cells [15]. Higher level of 5-HT2AR targeting autoantibodies in the lean heterozygote Zucker rat supports a role for upregulated adaptive humoral immunity (in part) due to a heterozygous leptin receptor mutation causing hyperleptinemia. More study is needed to determine whether high titer of autoantibodies in the lean heterozygous Zucker rat may predispose to immune complex formation that could bias signaling pathway activation downstream of 5-HT2A receptor binding.

In summary, 5-HT2A receptor autoantibodies increased in the circulation in male hypertensive ZDF rat between 8.5-10-weeks of age and persisted up until 25-weeks old or longer. The ZDF rat 5-HT2A receptor-targeting autoantibodies caused acute neurite retraction and accelerated mouse neuroblastoma cell loss by a mechanism involving activation of Gq11/PLC/IP3R pathway and RhoA/Rho kinase coupled signaling. The ZDF and lean heterozygous Zucker rat strains may be suitable animal models for investigations of the roles of obesity-associated inflammation and dysregulated humoral immunity, respectively, in the etiology of certain forms of late-onset neurodegeneration.

Funding

This project was supported by grants from the New Jersey Commission on Brain Injury Research (CBIR19PIL007) to MBZ and the Biomedical Laboratory Research and Development Service of the VA Office of Research and Development (I1BX004561) to KCHP. The views expressed in this article are those of the authors and do not necessarily reflect the position or policy of the Department of Veterans Affairs or the United States government.

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DOI: 10.31038/EDMJ.2020434

Abstract

Aim: To study the clinical and metabolic characteristics of newly diagnosed type 2 diabetes (T2DM) in urban clinics (CDD) and also to compare with the screen detected new diabetes cases (SDD) during an urban population survey.

Methods: Newly diagnosed T2DM (aged 20-60 years, n=741), based on blood glucose and Glycosylated haemoglobin (HbA1c) of ≥6.5% (48 mmol/mol) were selected. Demography, anthropometry, blood pressure, glycaemic and lipids profiles were analysed. Relevant statistical tests were used for group comparisons.

Results: Both groups had young age (45.0 ± 8.6 years) at diagnosis. Fasting blood glucose (p<0.05) and HbA1c (p<0.0001) were higher in CDD. Mean values of HbA1c were 9.1 ± 2.3% (76 ± 20 mmol/mol) in CDD and 8.3 ± 2.4% (67 ± 19 mmol/mol) in SDD (p<0.0001). Values  of HbA1c were higher   than ≥9.0% (75 mmol/mol) in 44.6% of CDD versus 26.4% of SDD (z=4.60, p<0.0001). SDD had higher body mass index (p<0.0001), abdominal obesity (p<0.005), hypertension (p<0.0001), cholesterol (p<0.005) and low density lipoprotein cholesterol (p<0.05) than CDD.

Conclusion: Both groups had young age at diagnosis. CDD had more severe glycaemia than SDD, probably suggesting that the clinic visits were delayed and therefore had longer period of undiagnosed diabetes. In comparison to CDD, SDD had higher metabolic abnormalities although the HbA1c values were lower.

Keywords

Newly diagnosed type 2 diabetes, South Asians, Clinical characteristics, HbA1c, Metabolic abnormalities

Introduction

Many developing countries show a rising trend in the prevalence and also in the incidence of type 2 diabetes T2DM [1-3]. Recently, a systematic review of studies on trends in the incidence of T2DM among adults in developed countries has shown that between 2006 and 2014, 27% of the reported populations had a stable incidence over time, while 36% reported a declining trend and 36% reported an increasing trend in the incidence of T2DM [4]. The declining trend in the incidence has been shown mostly in the developed countries. A huge clinical burden of newly diagnosed T2DM is present in developing countries. There is only limited real-world data describing the clinical characteristics of such patients [5].

The prevalence of diabetes is increasing rapidly in India, which is estimated to be 77 million adults in 2019 [1]. A recent epidemiological study done in urban India showed that, the prevalence and incidence of diabetes have increased significantly in all areas, including the Peri Urban Villages (PUV) of Tamil Nadu [2]. Using the data from the two studies conducted in 2006 [6] and 2016 [2] to estimate the incidence of diabetes, it was noted that, a sharper increase in the incidence occurred in the urban areas when compared with the villages [2]. A similar observation was made in the INdia DIABetes (INDIAB) study conducted in 15 states of India [7] as well as in a cohort study of 10 years follow-up in the urban area in southern India [8].

Several peculiar features are seen among the newly diagnosed South Asian populations with T2DM such as, younger age at onset, delayed clinical diagnosis due to lack of public awareness regarding the disease, reluctance to undergo periodic medical check-up and a long asymptomatic phase of T2DM [3,9]. The diagnosis of diabetes is often delayed, more so in the developing societies. Previous studies have described the characteristics of patients attending the clinics, who are under treatment [3,6,8]. There is sparse data on the profile of clinically diagnosed new T2DM patients in India.

The aim of this project was to study the clinical and metabolic characteristics of newly diagnosed adult T2DM patients from different clinics (CDD). We compared the profile with that of new T2DM identified during an epidemiological screening of urban population (SDD) [2].

Methods and Materials

Study Subjects

This is a study of newly diagnosed adult T2DM from 12 different centres located in four southern states of India. Patients with a previous history of diabetes or had taken anti-diabetes treatment were excluded from the study by the clinicians. Persons with prediabetes were also excluded. Details of men and women in the selected age group, who were treatment naïve were collected and analysed. For comparison, a group of newly detected T2DM subjects from an epidemiological survey of the urban population was taken.

All participants reported on fasting on the day of testing. In the clinics, the diagnosis of diabetes was based on blood glucose (fasting blood glucose ≥7.0 mmol/l and/2 hour Post Prandial Glucose (PPG)≥11.1 mmol/l) [10]. In the epidemiological study, diagnosis was made based on fasting and 2 h Post Blood Glucose (PBG) values. The 2 hour PBG was measured after giving 75 g of glucose load. We selected only patients who had an HbA1c value of ≥6.5% (48 mmol/mol) [11]. The total number of subjects analysed was 741, comprising of 514 from CDD and 227 from SDD; Identifier: NCT03490136 [2].

All centres followed uniform and standardised procedures for metabolic and clinical assessments. The study details and methodology were discussed in a meeting of the investigators, participating physicians and the researchers prior to the commencement of the study.

The Ethics Committee of the India Diabetes Research Foundation and Dr. A. Ramachandran’s Diabetes Hospitals approved the study. A written informed consent was obtained from all participants prior to the enrollment, at all centres. It was also agreed that the identity of the participants would not be disclosed. All study procedures were carried out in accordance to the ethical guidelines.

Clinical Assessments

Age, sex and presence of family history of diabetes were collected. Height was measured to the nearest centimeter using a stadiometer with the patient standing erect. Weight was measured to the nearest 0.1 kg using a digital weighing scale. Body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters. Generalized obesity was defined as BMI ≥23.0 kg/m²., BMI between ≥23.0 and 24.9 kg/m² was considered overweight, and BMI≥25.0 kg/m² was defined as obese. Waist circumference (WC), the smallest girth between the coastal margin and the iliac crest, was measured. Abdominal obesity was indicated by WC of ≥90 cm for men and ≥80 cm for women. Blood pressure was measured in the sitting position using the electronic measuring device (Omron HEM 7132; Omron, Tokyo, Japan). An average of two readings taken at 5-minutes’ interval was recorded. Persons with a history of hypertension and those with newly diagnosed diabetes with blood pressure readings ≥140/90 mmHg were categorized as hypertensive. All the clinical and metabolic details were recorded in a standardised questionnaire.

Biochemical Assessments

Glucose was measured in the respective study centres by the glucose oxidase method. Other biochemical parameters such as HbA1c and lipid profile were estimated at the central laboratory (Dr. A. Ramachandran’s Diabetes Hospitals, Chennai). Glycosylated haemoglobin (HbA1c) was measured by immunoturbidimetry (TINA-QUANT II; Roche Diagnostics Corporation, Germany); a procedure certified by the National Glycohemoglobin Standardization Program. Fasting serum lipid profile was estimated by standard enzymatic procedures using the reagents of Roche Diagnostics, Germany. HDLc was estimated by the direct assay. Samples were shipped to the central laboratory between 2° and 8°C on the same day of collection.

Statistical Analyses

Data are presented as mean ± standard deviation (SD) for continuous variables with a normal distribution, as median with interquartile range for skewed continuous variables and as number and frequency (%) for categorical variables. Independent samples t test, chi-square or Z – test were used to assess group differences for continuous variables and categorical variables respectively. Mann Whitney U test was used for skewed variables. A p value of <0.05 was considered statistically significant. Statistical analyses were performed using SPSS version 21.0.

Results

Table 1 shows the demographic and anthropometric characteristics of the two groups with newly diagnosed T2DM. CDD had higher percentage of men than SDD (p<0.05). Both groups had similar mean age at diagnosis (45.2 ± 8 years). The mean BMI was higher in SDD (p<0.0001). Prevalence of overweight and obesity were similar in both the groups. Women in SDD had higher WC (p<0.005) and the percentage of abdominal obesity (≥80 cm) was also higher in SDD (p<0.05). SDD had higher mean diastolic blood pressure (p<0.0001) and higher percentage of hypertension (p<0.005).

Data are presented as mean ± SD for continuous variables with normal distribution and as frequency (%) for categorical variables. Independent samples t test and chi-square test or Z test were used to test inter group differences for continuous variables and categorical variables.
CDD vs.SDD=^*p< 0.05; ^#p< 0.005; ^$p< 0.0001.
CDD, Clinically Diagnosed Diabetes; SDD, Screen Detected Diabetes; BMI, Body Mass Index; BP, Blood Pressure.

Table 2 shows the metabolic profile of the study groups. SDD had lower FBG (p<0.05) and HbA1c (p<0.0001) when compared with CDD. Among the total cohort of 741, 445 (60.1%) had HbA1c values
≥8.0% (64 mmol/mol), of which 289 (39.0%) had values ≥9.0% (75 mmol/mol).

Data are presented as mean ± SD for continuous variables with a normal distribution, as median with interquartile range for skewedcontinuous variables and as frequency (%) for categorical variables. Independent samples t test and chi-square test or Z testwere usedto test intergroup differences for continuous variables and categorical variables. Mann Whitney U test was used for skewed variables.
CDD vs.SDD=^*p<0.05; ^#p<0.005; ^$p<0.0001
CDD: Clinically Diagnosed Diabetes; SDD: Screen Detected Diabetes; HbA1c: Glycosylated hemoglobin; LDLc: Low Density Lipoprotein Cholesterol; HDLc:HighDensity Lipoprotein Cholesterol.

It was observed that a larger proportion of CDD (67.9%) had values of ≥8.0% (64 mmol/mol) versus 42.3% of SDD (z=6.47, p<0.0001). HbA1c values ≥9.0% (75 mmol/mol) were present in 44.6% in CDD versus 26.4% in SDD (z=4.60, p<0.0001). Among the lipid variables, total cholesterol (p<0.005) and Low Density Lipoprotein cholesterol (LDLc) (p<0.05) were higher in SDD.

Discussion

There have been many reports highlighting the differences in the clinical profile of diabetes between South Asians and the western populations [12,13]. But, there is sparse data on the clinical and metabolic characteristics of newly diagnosed T2DM from India. In this communication we report the clinical and metabolic characteristics of newly diagnosed diabetes patients recruited from different centres in southern India (CDD). The group represents the urban population with diabetes. We compared its clinical profile with a group of undiagnosed diabetes detected during an urban epidemiological survey (SDD).

In our study, the mean age of the newly diagnosed T2DM in both the groups was similar (45.2 ± 8.6 and 45.0 ± 8.6 years in CDD and SDD respectively). Another large, multicentre, cross-sectional study among diabetes patients in India also showed that the mean age at onset of T2DM was 45.4 ± 10.9 years which was similar to the observation made in our study [14]. This was significantly lower than that reported among the newly diagnosed T2DM cases in the United States (55.6 ± 13 years) [5]. Similarly, in two large multi-ethnic studies from UK [15,16], the age at diagnosis of T2DM was found to be lower (51.5 ± 10.42 and 52.6 ± 13.5 years respectively) in the South Asian population when compared to the White population (58.9 ± 10.09 and 63.3 ± 13.8 years respectively). South Asians develop T2DM at least a decade earlier.

Lower BMI with higher HbA1c in the CDD compared to SDD could possibly be due to a reduction in weight usually associated with moderate to high levels of undetected hyperglycaemia. In an earlier study, we had observed that more than 20% of the cases had weight loss prior to the clinical diagnosis of T2DM [17]. Among the Asian populations, Indians have a reluctance to undergo regular health check-up and medical consultation is often sought only when the symptoms or related problems arise due to diabetes [9]. Similar observation had been reported earlier in migrant Asian populations in western countries [15,18]. In the UK, a multiethnic South London study mentioned above showed that HbA1c levels were higher in South Asians than in the Europeans at an early period after the diagnosis. This was partially attributed to a delayed diagnosis in the Asians [15]. In our study, the significantly higher FBG and HbA1c observed in CDD could be related to a delay in the diagnosis of diabetes when compared with those diagnosed during an epidemiological screening. There is a higher likelihood of a few CDD patients having taken treatment for hypertension and hyperlipidaemia prior to visiting a diabetes clinic.

In our study, 44.6% of the patients had significantly higher HbA1c ≥9.0% (75 mmol/mol) when compared with 21.7% of the newly diagnosed patients in the clinics in USA [5]. There may be a number of reasons for high HbA1c in newly detected diabetes. One important reason could be longer periods of undetected T2DM in our population, partially due to the delay on the part of the patients to seek medical help. In India, a larger percentage of men report for medical consultation than women. Therefore, the proportion of men was more among the clinically diagnosed cases.

In an earlier study, we had noted that T2DM remains asymptomatic for a longer period of time and many develop complications such as retinopathy even before diagnosis [19]. We did not record the presence of symptoms or complications in this study.

Hypercholestremia seen among the newly diagnosed patients may also be related to the delayed diagnosis of diabetes and higher blood glucose levels. In SDD, screening for diabetes was done among adults with neither a previous history nor the presence of symptoms of diabetes. There is a higher chance of early detection of diabetes among this group than in the clinic diagnosed cases. The likelihood of early detection of hypertension is also higher when screening is done.

Our study describes the clinical and metabolic features of newly diagnosed T2DM in the clinics. The differences with the screen detected diabetes in the population probably indicate late diagnosis in the clinics.

Declarations

Funding

The study was funded by India Diabetes Research Foundation, Chennai.

Conflict of interest

None

Ethics approval

The study was approved by the Ethics Committee of the India Diabetes Research Foundation and Dr. A. Ramachandran’s Diabetes Hospitals.

Consent to participate

A written informed consent was obtained from all participants prior to the enrollment in the respective studies.

Authors’ contributions

Arun R, AR, CS, AN and RV contributed to the study design, developed the protocol, supervised the study, drafted the manuscript and revised it with critical input. AR, CS, Arun R, PS and KS contributed to data preparation and analyses. KS and PS participated and coordinated the field work and data collection. All authors have read and approved the final draft of the manuscript.

Acknowledgements

The authors acknowledge the support rendered by the study physicians of various hospitals for providing the data source of the clinic patients. The help rendered by Mr.M.Karthikeyan and the epidemiology team of the India Diabetes Research Foundation in coordinating the epidemiological screening is greatly acknowledged. We are grateful to all the participants of the study for their co- operation and support.

Abbreviation

BMI: Body Mass Index

CDD: Clinically Diagnosed Diabetes

FBG: Fasting Blood Glucose

HbA1c: Glycosylated Haemoglobin

PBG: Post Blood Glucose

PPG: Post Prandial Blood Glucose

SDD: Screen Detected Diabetes

T2DM: Type 2 Diabetes

WC: Waist Circumference

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