Assessment of Serum Resistin: A Recently Identified Adipocyte-Derived Hormone in Obese and Lean Individuals

Introduction

Obesity, with its growing occurrence and prevalence, stands as the most widespread metabolic condition affecting people worldwide. Overweight individuals make up more than half of the global populace. In addition to insulin resistance (IR), dyslipidemia, and type 2 diabetes mellitus (T2DM), there is a growing global epidemic now underway. Thus, a concomitant increased trend in diabetes mellitus (DM) data has been noted in tandem with an astounding increase in obesity. The chance of developing diabetes rises by 4.5 to 9% for each kilogram of weight gain. According to estimates, there were 200 million obese people in the globe in 1995. This number was 315 million after seven years. The World Health Organization has thus termed the increased prevalence of obesity and diabetes as a ‘21st Century epidemic’.¹

Low-grade systemic inflammation is a hallmark of obesity. Research on the role of adipose tissue as an active participant in directing the body's physiologic and pathologic processes has increased dramatically in recent years due to epidemiological facts about the rising tide of obesity and related illnesses. A wide range of adipocyte-derived factors, or adipocytokines, are produced by adipose tissue, an active endocrine organ. These factors include resistin, adiponectin, leptin, interleukin-6, and tumor necrosis factor-α. Adipocytokines act through autocrine, paracrine, and endocrine pathways, and their effects are varied.²

Insulin resistance is greatly influenced by resistin, one of the newly discovered adipocyte-derived hormones.² Three separate groups employed a range of methods to detect resistin.^2,3,4

Using thiazolidinediones (TZDs) as a trigger, Steppan et al. (2001) screened genes that were activated during fat cell differentiation but became down-regulated in mature adipocytes. This resulted in the identification of a polypeptide that the researchers called ‘‘resistin’’ (for insulin resistance). A conserved pattern of 11 cysteine residues at the C-terminal end of the structure is shared by resistin, often referred to as resistin-like molecules (RELMs), one of a family of three proteins.² As part of a recent study on molecules linked to allergic inflammation and airway hyperresponsiveness (AWH), the term FIZZ (found in inflammatory zone) was discovered. In three mouse genes and two human homologs (mFIZZ1, mFIZZ2, mFIZZ3, hFIZZ1, and hFIZZ3) make up the FIZZ gene family. According to Holcomb et al. (2000), the mFIZZ3 polypeptide was only expressed in white adipose tissue. Synonyms for the protein resistin include adipocyte-specific secreted factor (ADSF) and found in inflammatory zone (FIZZ3), which together make up the current nomenclature.^2,3,4

Human peripheral blood contains oligomerized resistin molecules in a variety of low and high-molecular-weight isoforms. In macrophages and monocytes, resistin's oligomeric and dimeric forms can both activate TNF-α and IL-12. However, the inflammatory effect of the resistin is unaffected by its structure.^5,6

Resistin up-regulation has been linked to diabetes, insulin resistance, and obesity, according to numerous research. Moreover, periodontal disorders and rheumatoid arthritis both have markedly higher serum levels of resistin. When combined, these data point to a potential function for this cytokine in the relationship between diabetes and obesity.^7,8

Discovering the most sensitive and specific biomarker is becoming more and more possible with the development of technologies for early illness detection, intervention, and fast treatment. Any disease can be detected using serum as a biomarker. Resistin is one such recently identified serological marker with strong biomarker properties.⁹

Materials and Methods

Fifteen obese, non-diabetic individuals with postprandial blood sugar (PPBS) <140 mg/dl, (Ppbs range should be below 160 mg/dl, so our sample contains non diabetic range below 160 mg/dl), body mass index (BMI) >30±5 and high-density cholesterol level (HDL) <60 mg/dl, low-density cholesterol level (LDL) >100 mg/dl, very low-density cholesterol level (VLDL) >30 mg/dl as entered in their case records were selected from the Endocrinology out-patient department, Narayana Super Specialty Hospital and classified under Group-I. Age (25-40 years) and sex-matched healthy non-diabetic individuals were chosen from the out-patient pool of the Department of Oral Medicine and were assessed for PPBS, BMI, HDL, LDL, VLDL. Only fifteen individuals whose PPBS values <140 mg /dl, BMI <20 ± 2, HDL >60 mg/dl, LDL <100 mg/dl, VLDL <30 mg/ dl were included in the study under Group II (Healthy controls) (HC). All Individuals of both groups belonged to the same race, ethnicity, socio-economic status, and living standards.

The individuals diagnosed with diabetes, along with any associated problems (retinopathy, peripheral neuropathy, nephropathy, etc.), and those who had any other illnesses that would affect the immune system were excluded from the study.

Screening examination included

• The patients' medical and dental histories

• The individuals' anthropometric weight (kg) and height (m) measurements to determine their BMI (weight divided by the square of their height, kg/ m2 ). BMI ≥30 is considered obesity, BMI 25–29.9 is considered overweight, and BMI 20–24.9 kg/m2 is considered normal weight.

Before the study began, all participants gave their written and verbal informed consent and the study methodology was described to them. Before getting blood samples, a case history was collected, paying special attention to details like age, sex, activity routine, and medications consumed. For each Group-I patient, information was taken from their individual endocrinology outpatient case records, including PPBS, BMI, LDL, HDL, and VLDL values.

Serum collection

By venipuncture, 4 ml of peripheral venous blood was extracted from the antecubital fossa using a 20-gauge needle and a 5-mL syringe. After that, the blood was quickly moved to the laboratory and placed in the vacuum-sealed tubes. After an hour of clotting at room temperature, the blood sample was centrifuged for 15 minutes at 3,000 rpm to extract the serum.

Postprandial blood sugar (PPBS)

The GOD/POD method (Trinder's approach, which uses glucose oxidase and peroxidase enzymes coupled with the chromogen 4-aminoatipyrine and phenol) was utilised to estimate the PPBS levels only for Group II participants. For use in additional tests, the residual serum was kept in Eppendorf tubes at -20ºC.

Serum resistin

Using the enzyme-linked immunosorbent assay technique, resistin levels were measured using the HUMAN RESISTIN ELISA KIT (Chongqing Biospes Co., Ltd., China; Catalogue No.: BEK1196).

ELISA

Pre-coated 96-well plates were filled with a polyclonal antibody against resistin. Additionally, as detecting antibodies, a polyclonal antibody against resistin coupled with biotin was employed. The wells were then filled with the standards, test samples, and biotin-conjugated detection antibody. A wash buffer was then used to wash wells. The unbound conjugates were then removed using a wash buffer solution after the addition of the Avidin-Biotin-Peroxidase Complex. Following that, the HRP enzymatic reaction was observed using TMB substrates. When acidic stop solution was added, the blue product that TMB had been catalysed by HRP to generate turned yellow. The density of yellow corresponds to the quantity of Resistin sample that was caught in the plate. Resistin concentration was computed after the OD absorbance in a microplate reader was measured at 450 nm.

Statistical analysis

IBM SPSS Version 22.0 was used for the descriptive statistics and analysis. The data values were represented as mean and standard deviation for continuous variables. The mean difference between the two groups was examined using the Student's T-test. To evaluate the correlation between the groups, Pearson's correlation test was taken into consideration. The p-value less than 0.05 was regarded as statistically significant.

Results

Relative to Group II (46.33±63.56 ng/ml, 48.93±3.59), Group I's serum resistin HDL mean levels were lower (11.09±38.09 ng/ml, 31.07±2.25 mg/dl). When comparing Group I and Group II, a very high significance (p<0.0001) was found in HDL, but an inconsequential p-value of 0.78 was found in serum resistin. Between the two groups, there was a weak p-value of 0.441 and a negative correlation (-.215) between the serum resistin levels. A statistically significant difference (p-value <0.0001) was seen in the mean BMI, LDL, and VLDL values between Group I and Group II (HC) participants.

The mean serum resistin and HDL levels for each group are displayed in Figure I and Table I. Table II displays the p-values for HDL and serum resistin in each group. Table III displays the relationship between the two groups' serum resistin levels.

Group I showed higher mean values of BMI, LDL, and VDL (33.20 ± 1.42 mg/dl, 144.60 ± 9.06 mg/dl, 43.87 ± 3.64 mg/dl) compared to Group II (21.87±1.30 ng/ml, 102.60 ± 5.70 mg/dl, 21.13 ± 2.36 mg/dl), with a very significant difference (p-value of <0.0001) between the two groups.

Figure II displays the BMI for each group. Table I displays the average BMI, LDL, and VLDL values for the two groups.

Serum resistin in group I has a positive association (0.110) with HDL and a negative correlation (-0.398) with BMI, -0.263 with LDL, and -0.225 with VLDL. Serum resistin in group II, however, has a comparable negative connection with BMI (-0.398), LDL (-0.43), and VLDL (-0.30) and positive correlation with HDL (0.19).

Table 4 and 5 show the serum resistin concentration correlation with BMI, HDL, LDL, and VLDL in obese and lean individuals (Group I and II).

Discussion

The hallmark of obesity is a rise in adipose tissue, a major source of proinflammatory cytokines such resistin, TNF-α, IL, visfatin, and adiponectin. It has long been known that adults who are overweight or obese are more susceptible to developing a number of chronic inflammatory diseases and disorders, including diabetes, arthritis, and cardiovascular disease. The primary conclusions of this study indicate that lipid profiles and anthropometric parameters differ in obese persons with respect to resistin levels.⁹

According to preliminary research, obesity in mice with IR is linked to higher levels of circulating resistin.² As one of the three murine resistin isoforms is absent from humans and resistin's homology with mice is imperfect, resistin's physiological function in humans may differ from that of mice. There is disagreement concerning resistin's function in the metabolic parameters as well. Resistin levels in humans have been linked to insulin resistance and obesity, according to some authors, although resistin levels and metabolic indicators have not been linked in any studies.^2,7

It was discovered that there may be a connection between resistin, diabetes, and obesity in rodents. For example, it was shown that diet-induced and hereditary variants of obesity in mice resulted in higher levels of circulating resistin. These levels were then neutralised by administering anti-resistin, which subsequently improved insulin action and blood glucose levels. On the other hand, giving recombinant resistin to normal mice will result in decreased insulin action and glucose tolerance. Furthermore, anti-diabetic medications (TZDs) lower resistin levels, supporting resistin's possible involvement as a mediator between obesity and diabetes.²

A causal role for resistin in glucose homeostasis has been demonstrated by research using animal models with changed serum resistin levels. For example, resistin gene-deleted mice had lower blood glucose levels following fasting because their livers produced less glucose than normal. This shows that resistin plays a substantial role in the hyperglycemia associated with obesity.

Many attempts have been made to explain why resistin is so important to humans. However, there is ongoing debate regarding resistin's precise function in human illness. Numerous investigations found that elevated human resistin levels were linked to diabetes, IR, or obesity. On the other hand, some published research reveals inconsistent findings and was unable to discover this kind of correlation.^7,10

Although resistin was initially linked to the onset of insulin resistance and diabetes in humans, there is an ongoing debate on its precise involvement in obesity, insulin resistance, and the etiology of type 2 diabetes. Steppan and colleagues made several significant discoveries when resistin was initially discovered in 2001. In animals with hereditary and diet-induced obesity, resistin levels were initially higher. Moreover, the administration of anti-resistin antibodies to obese and insulin-resistant mice enhanced their responsiveness to insulin. Furthermore, in healthy mice, recombinant resistin therapy reduced insulin action and glucose tolerance. Finally, injection of resistin impeded adipocytes' ability to absorb glucose generated by insulin. Based on these results, it was shown that resistin is essential for obesity and insulin resistance in the diabetic mice model. However, it is difficult to say how far these explanations can be carried over into human investigations.²

Recent research has produced a wealth of data supporting resistin's critical involvement in obesity and insulin resistance. It has been established that resistin causes IR and is expressed in human hepatocytes and hepatic tissues. Resistin mRNA levels were also readily found in human peripheral blood mononuclear cells (PBMCs) and were found to be greater in female type 2 diabetics than in healthy women, indicating that resistin may have a role in the pathophysiology of type 2 diabetes in humans.

Resistin has been linked to human obesity, insulin resistance, or diabetes, according to certain research.^7,11 This type of correlation was not discovered in other published investigations. In the end, there hasn't been much consensus regarding resistin's precise function in those human diseases.^10,12

Numerous investigations have revealed that the amount of serum resistin was considerably greater in obese persons compared to lean individuals.^7,13 On the other hand, lean, healthy people and obese people did not significantly differ in their serum resistin levels, according to other research.¹²

It has been discovered that there is a positive correlation between serum resistin levels and BMI, waist circumference (WC), waist-to-hip ratio (WHR), homeostatic model assessment for insulin resistance (HOMA-IR) index, serum insulin, plasma glucose, and HbA1c levels.^7,12-17 However, some research found a negative correlation or no correlation at all between resistin levels and BMI, WC, WHR, HOMA-IR, fasting plasma glucose, or insulin levels.^11,18,19

Diverse investigations were conducted to ascertain the levels of resistin in circulation and its correlation with type 2 diabetes, insulin resistance, and obesity. The findings were inconsistent, with some studies showing a substantial relationship between serum resistin levels and T2DM, IR, and obesity, while others did not. These differences could have a variety of causes, such as the use of different assay techniques, low enrollment numbers in various research, and variations in the demographics of the study groups.

Summary of studies showing increased resistin levels in T2DM, IR and obesity

Summary of studies decreased resistin levels in T2DM, IR and obesity

Conclusion

In obese people, circulating resistin concentration is linked to both HDL cholesterol and anthropometric measurements. Additional research is required to examine this ambiguous subject with potential therapeutic and clinical ramifications for a wide range of patient types. In summary, there is still disagreement on the precise function of human resistin in obesity, insulin resistance, and the onset of diabetes. Soon, a clear role in metabolism for the intriguing novel hormone resistin will be discovered. This study is the ‘‘FIRST in the Indian Scenario,’’ comparing serum resistin levels in lean and obese people with the necessary parameters.

Conflict of Interest

Nil