Leptin/Adiponectin Ratio is Progressively Elevated in Pre-diabetic to Type 2 Diabetic African American Adults
Corresponding author:Dr. Kanwal K. Gambhir, Molecular Endocrinology Laboratory, Department of Medicine, Howard University College of Medicine, 2041 Georgia Ave, NW Suite 5C02, Washington, DC 20060, USA, Tel: 202-806-1398; Email: firstname.lastname@example.org
The number of people with diabetes mellitus and insulin resistance continues to rise year by year, and occurs at particularly high rates in middle aged and elderly people [1-3]. The likelihood of having diabetes varies between ethnicities, with African Americans twice as likely as Caucasians to have type II diabetes mellitus (T2DM) . Patients with T2DM can develop insulin resistance, a condition that is associated with abnormal levels of two hormones released by adipose tissue – adiponectin and leptin [5-13].
Adiponectin plays an important role in protecting against insulin resistance and other metabolic diseases [1, 14]. It does so by activating AMP-activated protein kinase (AMPK) in the liver and skeletal muscle, which in turn stimulates glucose use and fat combustion in these organs [15, 16]. However, studies show that patients with T2DM have decreased levels of adiponectin compared to non-diabetic patients likely due to suppression of adiponectin production by TNF-alpha, a hormone that is present in high levels in inflammatory states like T2DM [17,18]. Conversely, plasma leptin appears to increase in patients with T2DM and is directly proportional to insulin resistance [5-13]. Coimbra et al. conducted one of the studies that analyze the levels of these two adipokines in patients with T2DM . They found that patients with T2DM had significantly lower adiponectin and significantly higher leptin levels compared to control patients without T2DM . They also found that female patients had higher leptin levels than males regardless of whether they had T2DM or not . Moreover, age influenced adiponectin and leptin levels, with leptin levels increasing and adiponectin levels decreasing with age . An interesting finding of this study was a link between the length of disease and hormone levels . Statistical significance was lost when comparing adiponectin levels between control and T2DM patients after adjusting for length of disease, and the same occurred for leptin levels in female patients .
Other studies analyzing adipokine levels in T2DM patients include those conducted by Hung et al. who studied at 1094 patients in Australia, and Mojiminiyi et al. who studied 135 patients in Kuwait [6, 7]. Mojiminiyi et al. showed that leptin levels increased with obesity, insulin resistance, and CRP levels while adiponectin trended in the opposite direction . The study by Hung et al. focused on adiponectin levels and showed an inverse relation to BMI, waist-hip ratio, diastolic blood pressure, triglycerides, blood glucose, and fasting insulin levels . They also showed a positive correlation of adiponectin with HDL cholesterol . Importantly, the association between adiponectin levels and insulin sensitivity was seen even in patients with normal BMI, suggesting that patients need not have high adiposity in order to encounter lower adiponectin levels . These findings lend to the belief that adiponectin is a key player in the development of metabolic syndrome and its components . This study did show that adiponectin levels in some insulin sensitive patients were low, a contradictory trend that has been noted by some other studies as well [19, 20]. This suggests that hormones other than insulin may also be involved in dictating adiponectin levels .
While the inverse relationship of adiponectin and direct relationship of leptin to obesity, diabetes mellitus, and insulin resistance have been frequently noted, there appears to be some variation in the levels of these hormones between ethnicities [21, 22]. For example, one study investigating T2DM patients of White, Chinese, African American, and Hispanic ethnicity found that Chinese patients had the lowest plasma levels of adiponectin and leptin . They also found that adiponectin levels were highest in the Caucasian group while leptin levels were lowest in the African American group . Although several studies have investigated the relationship between T2DM and plasma adipokine levels, none to our knowledge have focused their investigation on minority groups in the United States. The goal of this study is to elucidate the effect of diabetes status on plasma leptin, plasma adiponectin, and leptin/adiponectin ratio (LAR) in African American adults with pre-diabetes or T2DM.
All components of this study are in compliance with guidelines of the Howard University Institutional Review Board (HUIRB). The HUIRB provided approval (IRB-13MED-73) of the protocol used to conduct the study. All participants in the study provided written informed consent.
The subjects in this study were previously recruited from the Diabetes Treatment Center (DTC) at Howard University Hospital. The present study consisted of 15 subjects with T2DM, 6 subjects with pre-diabetes, and 15 controls. The controls were healthy subjects who were recruited from within the Howard University community. Samples of whole blood were previously drawn from subjects after an 8-10 hour fasting period. T2DM subjects were previously diagnosed by a physician and patients at the DTC. T2DM was defined as subjects having a fasting blood glucose > 126 mg/dL of and HbA1C > 6.5. The subjects were all African American and between the ages of 18 and 80 years. All subjects were considered either high risk pre-diabetic or T2DM. In subjects with T2DM, the diagnosis was made prior to being enrolled in this study. Non-diabetic high-risk obese pre-diabetics were identified through the W.E.I.G.H.T. study at the DTC. These subjects were previously recruited based on a strong family history of T2DM and obesity.
Samples of venous blood were drawn and collected in heparinized vacutainer 6.0 mL tubes (BD Vacutainer Sodium Heparin 95 USP Units). Erythrocytes were isolated and then purified by the Hypaque-Ficoll gradient. The plasma layer was removed after initial centrifugation and stored at -30°C until use for hormonal assays
Hormone assays were conducted in the Molecular Endocrinology Laboratory at Howard University Hospital. Commercially available Millipore radioimmunoassay (RIA) kits were used to measure adiponectin and leptin concentrations following manufacture procedures. The leptin assay used 125I-labeled human leptin and a human anti-leptin antiserum in order to calculate the quantity of leptin present in the plasma and has 100% specificity for human leptin. The adiponectin assay used 125I-labeled murine adiponectin and a multi-species adiponectin rabbit antiserum to determine the level of adiponectin in the plasma. Adiponectin and leptin standards were prepared as per the manufacturer procedures by using serial dilutions. All samples were of 100 uL and were assayed in duplicate. The samples were counted using the Wallac 1470 Wizard Gamma Counter.
The quantity of unknown antigen was determined by extrapolating from a standard curve. These extrapolations were then used to determine the concentrations of hormone in each individual sample. The average values are expressed as mean ± standard error of mean. Student’s t-test or one-way ANOVA was used to determine statistical significance between two or three groups, respectively. The level of statistical significance was set at p ≤ 0.05.
The clinical characteristics of the patients in the study were previously reported by Fluitt et al. 2016 . The mean age of the patients in this study was 50.4 years. The age of the T2DM group mean age was 56.59 ± 2.06 years and the pre-diabetics group mean was 20.67 ± 0.56 years (p < 0.01). These groups were also both significantly different from the mean age of the control group, which was 31.47 ± 3.01 years. The T2DM group had a mean weight of 98.16 ± 5.14 kg, which was higher than that of the pre-diabetic group mean weight of 88.64 ± 8.55 kg (p=0.50). The control group had the lowest mean weight of 83.13 ± 4.56 kg, but this was not a statistically significant difference from the T2DM and pre-diabetic groups. The pre-diabetic group had a slightly higher mean BMI of 32.93 ± 3.99 kg/m2 compared to the T2DM group mean BMI of 31.59 ± 1.55 kg/m2. The control group had a lower mean BMI than both other groups, 28.22 ± 1.59 kg/m2, but none of these differences in BMI between groups was statistically significant. Random blood glucose was 204.1 mg/dL in the T2DM group. Random blood glucose was not measured in the high-risk prediabetic or control group. The T2DM group had a mean HbA1c of 8.62 ± 0.38, which was significantly higher than the pre-diabetic group mean HbA1c of 5.41 ± 0.14 (p < 0.01). HbA1c was not measured in the control group . In the T2DM group, HDL was 46.1 mg/dL and LDL 93.1 mg/dL. HDL and LDL were not measured in the pre-diabetic and control groups.
Plasma hormone concentrations
Plasma adiponectin and leptin concentrations were measured in samples obtained from non-diabetic, pre-diabetic, and T2DM African American patients. The plasma adiponectin was significantly lower in T2DM (2.52 ± 0.4 µg/mL, n=15) and pre-diabetic (4.18 ±1.4 µg/mL, n=6) patients compared to non-diabetic controls (19.1 ± 1.8 µg/mL, n=15) (Figure 1).
Figure 1. Plasma adiponectin concentration in non-diabetic, pre-diabetic and T2DM African American adults. Adiponectin concentration was measured using commercially available RIA kits in non-diabetic (n=15), pre-diabetic (n=6), and T2DM (n=15) African American Adults. One-way ANOVA with multiple comparisons was used to determine significance at p≤0.05.
Conversely, plasma leptin was higher in T2DM (49.4 ± 8.5 ng/mL, n=15) and pre-diabetic (74.0± 15 ng/mL, n=6) patients compared to non-diabetic controls (24.5 ± 12 ng/mL, n=9) (Figure 2).
Figure 2. Plasma leptin concentration in non-diabetic, pre-diabetic and T2DM African American adults. Leptin concentration was measured using commercially available RIA kits in non-diabetic (n=9), pre-diabetic (n=6), and T2DM (n=15) African American Adults. One-way ANOVA with multiple comparisons was used to determine significance at p≤0.05.
The Leptin:Adiponectin Ratio in African American Adults
The LAR was measured in all 3 groups for subjects with containing all data points for leptin and adiponectin. Subjects with missing data were removed from this analysis, leaving a total of 30 subjects (control n=9; pre-diabetic n=6; T2DM=15). The LAR was lowest in the control group and highest in the pre-diabetic group. The LAR was significantly higher in pre-diabetic (29.1 ± 12. ng/ µg, n=9) and T2DM (26.2 ± 5.2 ng/ µg, n=15) compared to non-diabetic controls (2.16 ± 1.2 ng/ µg, n=9) (Figure 3).
Figure 3. Leptin:Adiponectin Ratio in non-diabetic, pre-diabetic and T2DM African American adults. Leptin:Adiponectin ratio was calculated by using the following ratio: letpin (ng/mL) / adiponectin (µg/mL) in non-diabetic (n=9), pre-diabetic (n=6), and T2DM (n=15) African American Adults. One-way ANOVA with multiple comparisons was used to determine significance at p≤0.05.
Our study demonstrates that African American patients with pre-diabetes or T2DM have leptin and adiponectin concentration that vary significantly from healthy non-diabetic controls. Pre-diabetic and T2DM patients encounter an increase in plasma leptin and decrease in plasma adiponectin. Our study also shows that leptin and adiponectin levels do not vary much between diabetics and pre-diabetics in this ethnic specific cohort. Furthermore, we show that the LAR is significantly greater in T2DM patients compared to controls. While similar findings have been made in several earlier studies, to our knowledge this is the first study to investigate leptin and adiponectin levels, as well as LAR, specifically in African American T2DM and pre-diabetic patients. The importance of performing ethnicity specific studies on this topic stems from the variance in adipokine levels in T2DM patients of different ethnicities [21, 22].
We compared the plasma leptin and adiponectin levels in T2DM patients in our study to those seen in three randomly selected previous studies, and noted that patients in our study had a higher mean leptin and lower mean adiponectin compared to T2DM participants in these previous studies [1, 5, 6]. These studies were conducted in Portugal, Slovenia, and Kuwait and do not specify recruit participants of any specific ethnicity [1, 5, 6], suggesting that African American patients have an increased adipokine response to the diabetic state compared to patients of other ethnicities. A study conducted by Osei et al. investigated adiponectin levels in African American T2DM patients, and the mean adiponectin levels noted in this study were also lower than that seen in the randomly selected other studies mentioned above . Furthermore, a study conducted by Rasmussen-Torvik et al. investigated adipokine levels and LAR in Caucasian, Chinese, African American, and Hispanic patients . The investigators found that leptin levels were higher in the African American group compared to all other ethnicities in the study . They also found that adiponectin levels were lower in the African American group compared to the Caucasian and Hispanic groups, but higher than the Chinese group . This study provides further evidence that African American patients have an increased propensity for producing more leptin and less adiponectin in the diabetic state.
Considering that leptin and adiponectin are important players in impacting an individual’s sensitivity to insulin, it is important to consider the possibility that African American diabetic patients or healthy non-diabetic individuals with a strong family history of T2DM are more prone to decreased insulin sensitivity than people of other ethnicities. This finding was made by Haffner et al., whose study showed that African American patients with non-insulin-dependent diabetes mellitus (NIDDM) were less likely to be insulin sensitive than non-Hispanic white patients with NIDDM . However, the statistical significance of this difference was lost after adjusting for the greater obesity of the African American group . The study also showed no significant differences in fasting or 2-hour insulin levels between ethnic groups . An earlier study performed by the same group found that fasting and 2-hour insulin levels were both significantly higher among non-diabetic African Americans than in non-diabetic Caucasians . They also found that non-diabetic African Americans and more likely to be insulin resistant that non-diabetic Caucasians . These findings suggest a possible reason for the higher rates of NIDDM among African Americans compared to Caucasians .
It is currently known that the diabetic state has an impact on plasma levels of leptin and adiponectin, and by comparing the findings in our study to those of other studies, it appears that this impact occurs to a greater extent among African Americans than in some other ethnicities. The variance in leptin and adiponectin levels between ethnicities may be able to account for some of the differences in rates of T2DM and insulin resistance between ethnicities, but the full extent of their influence is yet to be elucidated.
A limitation of our novel pilot studies is the small sample size. Furthermore, there was a large difference in mean age between the three groups (control, pre-diabetes, and T2DM), with the T2DM group being the oldest and the pre-diabetic group being the youngest. This may have present confounding factors in establishing an association between a diabetic state and plasma adipokine levels. Future studies will include age- and sex-matched subjects to strengthen the current findings of this pilot study. Studies by Coimbra et al. and Cnop et al. found in their respective analyses that patient age was significantly and negatively correlated with adiponectin levels [1, 27]. Coimbra et al. also found that age was significantly and positively correlated with leptin levels . Another limitation of the study is that hormone levels for each patient were only measured once, not accounting for variations in hormone levels within individuals at different periods of time. However, these findings add to our current understanding of ethnic differences observed in insulin resistance/type 2 diabetes mellitus and may provide needed insight to develop personalized treatment options for persons of African descent. Moreover, this study supports the use of LAR as an important biomarker of T2DM. LAR may provide a parameter in diagnosing and assessing T2DM in African Americans.
The authors would like to acknowledge all the participants of this study, the Endocrine Fellows and Physicians of the Howard University Hospital Diabetes Treatment Center, and the phlebotomist that contributed to this study. The authors would also like to acknowledge the Nekhai Laboratory.
This work was supported by the JHU-UMD DRC Grant P30DK079637-08, Grant #4525, the NIH Research Grant 5G12MD007597 and partly by the Howard University Department of Medicine. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Authors declare no conflict of interest. KC drafted the manuscript and analyzed data, MBF, GNB, KKG designed study, performed experiments, recruited patients analyzed data and edited manuscript, and AD performed experiments and analyzed data.
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