Associations of Obesity-metabolic Status With Insulin Resistance and Chronic Inammation Level: Results From the CNTR Study

Background: Insulin resistance (IR) and inammation are the potential mechanism linking obesity and cardiometabolic risk. The aim of this work was to examine the joint relations of obesity and metabolic status with IR and chronic inammation level among Chinese adult twins. Methods: The analyses used data from 1113 adult twins in 4 provinces (Shandong, Zhejiang, Jiangsu and Sichuan) from Chinese National Twin Registry (CNTR) which collected detailed information. Those with 0 or 1 metabolic syndrome (MetS) components excluding waist circumference were considered metabolically healthy, and those with waist circumference ≥ 90 cm in men and ≥ 85 cm in women as obese. All participants were categorized into four phenotypes: metabolically healthy non-obesity (MHNO), metabolically healthy obesity (MHO), metabolically unhealthy non-obesity (MUNO), metabolically unhealthy obesity (MUO). High sensitivity C reactive protein (hsCRP) was measured to assess underlying inammation and homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as surrogate measure of insulin resistance. Results: In observational analyses of 1113 individuals (mean [SD] age, 46.6 [12.9] years; 463 obese [41.6%]). 20.3% obese twins were metabolic healthy. Serum HOMA-IR level was higher in MUNO (β=0.42, 95% CI: 0.21–0.64), MHO (β=0.68, 95% CI: 0.36–1.00) and MUO (β=0.69, 95% CI: 0.46– 0.91) twins, compared with their MHNO counterparts. The chronic inammation level, evaluated by hsCRP was similar between MHO and MUO, which differed signicantly to metabolic healthy non-obesity (MHNO). Within twin-pair analysis indicated there might exist common genetic inuence between HOMA-IR and MHO/MUO phenotype. Conclusions: Among Chinese adult twins, metabolic status were independently associated with higher IR while development of chronic inammation might closely relate to central obesity. It is necessary for the different risk assessment based on metabolic status in obese population. was for (smoking, obesity indicators (BMI, PBF, adjusted in the nal model. handled after logarithmic transformation in the regression analyses. Robust standard error and condence intervals for estimates have been produced. All the statistical analyses were performed with Stata statistical software (release Stata Corporation, TX). P-values are two-sided, and statistical signi ﬁ cance assumed at P<0.05. p < 0.001), and obese individuals with 3 MetS components (β = 0.88,95% CI: 0.65–1.12, p < 0.001) compared with those in non-obesity without any MetS component. With respect to levels of hsCRP, compared with those in non-obesity without any MetS component, participants with obesity and any MetS component had similar higher levels of hsCRP. the highest level was seen in participants with both obesity and 4 MetS components. Within twin-pair analysis indicated there might exist common genetic inuence between HOMA-IR and MHO/MUO phenotype. The chronic inammation level, evaluated by hsCRP was similar among obese twins with various number of MetS components, which differed signicantly to MHNO. This demonstrates that the development of chronic inammation might closely relate to central obesity but not MetS.


Introduction
Obesity is often associated with a constellation of metabolic abnormalities, including insulin resistance, impaired fasting glucose and/or tolerance, dyslipidemia, hypertension and metabolic syndrome (MetS), which are important risk factors for type 2 diabetes (T2D) and cardiovascular diseases (CVDs) (1,2). However, not all obese individuals entail metabolic abnormalities. The healthier phenotype displayed by some obese patients has been called metabolically healthy obesity (MHO). In general, the risks of T2D, CVDs, and all-cause mortality in those with MHO are lower than people with metabolic unhealthy obesity (MUO), but greater than in those who are metabolically healthy and non-obese (MHNO) (3)(4)(5)(6)(7), although inconsistent results have also been reported (8,9). Moreover, recent studies have found that the risks of these adverse outcomes are directly related to the number and severity of metabolic abnormalities (10)(11)(12)(13).
It is now generally accepted insulin resistance and in ammation are the potential mechanism linking obesity and cardiometabolic risk (14,15).
Insulin resistance is identi ed as an impaired biologic response to insulin stimulation of target tissues, primarily the liver, muscle, and adipose tissue. The metabolic consequences of insulin resistance can result in hyperglycemia, hypertension, dyslipidemia, visceral adiposity and contributing to cardiometabolic syndrome and increased CVD risk (16). Studies have found that insulin sensitivity is greater in people with MHO than in those with MUO, and many participants identi ed as having MHO are more insulin resistant than those who are MHNO (17)(18)(19).
Experimental and observational evidence also suggested that in ammation might play a central role in the pathogenesis of cardiovascular disease.
Some studies reported that MHO participants had signi cantly lower CRP levels than non-MHO (20,21). Karelis et al. reported that MHO women had a lower in ammatory state than women with postmenopausal insulin resistance and that this had a role in lowering the risk of cardiovascular disease (20). There are also studies found a similar level of CRP between two groups (22,23). To date inconsistent ndings were reported about the relationship between in ammation level and MHO/MUO phenotype.
The factors responsible for the greater preservation of insulin action in people with MHO than in those with MUO are not clear, but could be related to differences in potentially modi able lifestyle factors and genetic factors associated with adipose tissue biology. MHO is more often observed in young, physically active patients with a better nutritional status and low levels of ectopic and visceral fat storage (24). GWAS have identi ed genetic variants that are associated with increased adiposity in conjunction with a healthy metabolic pro le (25). As most previous studies were unable to control for the individual genetic variability it was unknown whether associations between metabolic abnormalities and clinical characteristics were attributable to shared genetic vulnerabilities in uencing both phenotypes. Twin design is seen as a useful method of controlling confounders in observational epidemiologic studies. Especially monozygotic (MZ) twins who are completely matched for any variations in the genetic background provide an extremely powerful control for genetic confounding factors.
Therefore, it is necessary to clarify the insulin resistance and chronic in ammation level of obese people that have signi cant metabolic abnormalities controlling genetic and familial factors. In this study, we aimed to investigate the joint relations of obesity and metabolic status with insulin resistance and in ammation in a large cohort of Chinese twin adults.

Methods
The participants belong to the Chinese National Twin Registry (CNTR), the rst and largest population-based twin registry in China described in detail elsewhere (26).
The analyses in this paper were based on a follow-up survey held from April to December 2013 among 1147 participants. The subjects were adult twins from four provinces covering 9 cities in Shandong, Zhejiang, Jiangsu and Sichuan province who completed an in-person questionnaire interview, a physical examination and a fasting blood biochemical test.
Pregnant female twins were excluded from participation. Twins were excluded from analyses if: (1) with a de nitive diagnosis of medical diseases such as alimentary tract tumor, cardiovascular heart disease, stroke and kidney disease; (2) treated with weight-lowering pharmacological agents. At last, a total of 1113 individuals (541 completed twin pairs and 31 individuals) were eligible for this study.
Determination of zygosity was based on the information from questionnaires during the baseline investigation. Twins of different genders were directly classi ed as DZ. For twins of the same gender, a model was built according to age, gender and 'whether they were as alike as two peas in a pod'. The model has been validated using DNA genotyping and found to be >90% accurate (27). All participants provided their written informed consent and Biomedical Ethics Committee at Peking University, Beijing, China approved the study protocol.

Clinical and Biochemical Data Collection
Data were collected with standardized computer-assisted personal interviews and medical examinations by trained staff. Information on demographic characteristics, medical history, and lifestyle factors were recorded, including questions on tobacco smoking (never, former, current), alcohol drinking (never, former, current) and exercise activities. Participants' exercise activities on occupation, transportation, daily life and leisure time were assigned a metabolic equivalent task (MET) value, using the Compendium of Physical Activities by Ainsworth et al. (28).
In addition, each participant's blood pressure, height, weight and percent body fat (PBF) were measured. Blood pressure was calculated as the mean of the second and third measurement out of three consecutive measurements. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared. Height was measured to the nearest 0.1 cm on a portable stadiometer while weight was measured to the nearest 0.1 kilograms using a digital balance (Body Composition Analyzer/Scale, TANITA, Tokyo, Japan). Waist circumference was measured three times at the level of the umbilicus to the nearest centimeter and the mean value was used in the analyses. PBF was determined by bioelectrical impedance (Body Composition Analyzer/Scale, TANITA).
Venous blood samples were collected, and serum total cholesterol (TC) and triglycerides (TG) were measured by the enzymatic colorimetric method (Roche, Basel, Switzerland). Direct methods were applied to assess high density lipoprotein (HDL) cholesterol and low density lipoprotein (LDL) cholesterol (Roche, Basel, Switzerland). A modi ed hexokinase enzymatic method was used to detect glucose (Glu) (Roche, Basel, Switzerland), and serum insulin was measured by chemiluminescence immunoassay (CLIA) on the ADVIA Centaur immunoassay system. Insulin resistance was estimated according to homeostasis model assessment (HOMA-IR): HOMA-IR = [fasting glucose (mmol/l) × insulin (U/ml)]/22.5. Serum highsensitivity CRP (hsCRP) was measured using a high-sensitivity immunoturbidimetric method (CRP [Latex] HS, Roche, Mannheim, Germany) on a Hitachi auto-analyzer (Roche Diagnostics, Mannheim, Germany). To minimize the effects of assay variability, samples from each twin pair were analyzed using the same assay.

De nition of the Phenotypes
We considered four components of the metabolic syndrome: 1) systolic BP ≥130 mmHg or diastolic BP ≥85 mmHg or self-reported hypertension or using antihypertensive drugs; 2) serum fasting glucose ≥5.6 mmol/L or self-reported diabetes or intake of antidiabetic medication; 3) HDL cholesterol <1.0 mmol/L for men and <1.3 mmol/L for women or using lipid-lowing drugs; and 4) triglycerides ≥1.7 mmol/L or using lipid-lowing drugs.. Individuals with waist circumference ≥90 cm (men) and 85 cm (women) were considered obese (29). According to the NCEP-ATP III criteria (30), participants with ≤1 abnormal component excluding waist circumference were de ned as metabolically healthy (MH), with the remaining de ned as metabolically unhealthy (MU). Status of metabolic health and obesity categories were combined to create the four phenotypes: metabolically healthy non-obesity (MHNO), metabolically healthy obesity (MHO), metabolically unhealthy non-obesity (MUNO), metabolically unhealthy obesity (MUO).

Statistical Methods
Data are presented as mean ± SD or median ( rst quartile (Q1) -third quartile (Q3)) for continuous variables and absolute and relative frequencies for categorical variables. We compared epidemiological, physical and biochemical characteristics between MHO and MUO twins as well as MHNO and MUNO twins. P values were corrected for the correlation between co-twins using generalized estimating equations.
Mixed-effect linear regression models with a random intercept for each twin pair to account for twin clustering were performed to examine the relationship of serum HOMA-IR and hsCRP levels with metabolic status (MH as reference group), number of MetS components, and the combined obesity-metabolic categories (MHNO as reference group),with adjustment for potential covariates. The rst model was adjusted for age, sex, place, and zygosity; the second model was additionally adjusted for lifestyle factors (smoking, drinking, and MET level), obesity indicators (BMI, PBF, WHR) were further adjusted in the nal model.
To investigate whether these associations were confounded by shared genetic and environmental factors, we applied co-twin regression analyses within twin pairs strati ed by zygosity. The within-pair approach automatically takes into account shared familial and environmental in uences.
Fixed effect models were used to estimate the relation of serum HOMA-IR and hsCRP levels with metabolic status (MH as reference group), number of MetS components and the combined obesity-metabolic categories (MHNO as reference group) separately for DZ and MZ twins adjusted for lifestyle factors (smoking, drinking, and physical activity) and obesity indicators (BMI, PBF, WHR).
All the serum metabolites were handled after logarithmic transformation in the regression analyses. Robust standard error and con dence intervals for estimates have been produced. All the statistical analyses were performed with Stata statistical software (release 12.0; Stata Corporation, College Station, TX). P-values are two-sided, and statistical significance was assumed at P<0.05. The MHO individuals had a mean age of 40.85 ± 12.03 years with a high proportion of women (52.1%). When this group was compared to the MUO phenotype, differences were found in lower WC, WHR (p < 0.0001) and lower concentrations of most biochemical characteristics except hsCRP.

Sample characteristics
Besides, the MHO group had the lowest proportion of current smokers (18.1%) and current drinkers (16.0%) and highest reported MET levels among four groups. Compared to MUNO, MHNO individuals were younger, with a higher proportion of women (48.9%), and were less likely to smoke and drink. Numerical data are presented as means (SD) or median (IQR), and categorical variables are presented as numbers (%).
Further, we analyzed the associations between MetS components and serum HOMA-IR and hsCRP levels. Individuals with 2 or more MetS components had a signi cantly higher level of HOMA-IR than normal individuals and a dose-response relation was found between numbers of MetS components and serum HOMA-IR after adjusting for obesity indicators and other covariates (P for trend < 0.001, Table S1). The association between numbers of MetS components and serum hsCRP began no more signi cant in the model additionally adjusted for obesity indicators. Strati ed analysis according to obesity status indicated that serum hsCRP was related to number of MetS components only in obesity twins (Table S2 and   S3).

Obesity-metabolic categories with insulin resistance, and chronic in ammation level
When combined metabolic status with obesity status, compared to MHNO individuals, the MUO group where both obesity and metabolic abnormalities were present, was characterized with the highest measurements for both indicators, followed by the MHO group. The MUNO group re ecting individuals with absence of obesity, but presence of metabolic abnormalities, was associated with the minimum increase in serum HOMA-IR and hsCRP levels. After adjusted for lifestyle factors (smoking, drinking, and physical activity), TC and HbA1c (model 3), all the associations were still signi cant thought there were a slight decrease (Table 3).

Within twin-pair analysis
In analyses controlling for genetic and familial effects within 541 complete twin pairs, associations of metabolic status with serum HOMA-IR and hsCRP levels are presented in Table 4. Compared to MH group, the MU group had signi cantly higher levels of HOMA-IR in both MZ and DZ twin-pair analysis adjusting for obesity indicators and other covariates. Besides, we analyzed the independent associations between MetS components with serum HOMA-IR and hsCRP levels. The results showed that compared with twins without any MetS component, level of serum HOMA-IR were signi cantly higher in twins with 4 MetS components in both MZ and DZ twin-pair analysis. Among obesity group, when compared with obese twins without any MetS component, obese twins with 4 MetS components had signi cant higher level of serum HOMA-IR(β = 1.01,95% CI: 0.07-1.96, p = 0.036,S4 Table).  Results of obesity-metabolic categories with insulin resistance, and chronic in ammation level were shown in Table 5 (31). Obesity is a wellestablished risk factor for IR, and previous studies indicated IR plays a major role in the pathogenesis of cardiometabolic disorders and is a common consequence of ectopic accumulation of visceral fat and intracellular lipid (32)(33)(34). When adjusted for obesity indicators, the association between metabolic status and insulin resistance attenuated, but remained statistically signi cant. Besides, we found the number of MetS components correlated with IR regardless of obesity status. All these results indicated that there exist an independent relationship between MetS and insulin resistance besides the effect of obesity.
In the past decade it has become increasingly clear that persons with similar fat mass may present with completely distinct clinical metabolic pro les (35,36). Similarly, we found a higher HOMA-IR levels of MUO with MHO group. This was consistent to previous studies conducted in obese adults (23,(37)(38)(39). In a study of 1458 adults from two independent populations, individuals with MUO had signi cantly higher level of HOMA-IR than MHO group (23). Another cross-sectional study of 3844 Spanish Caucasian adults showed the same results (39). The prevalence of MHO in our cohort of obese twins was 20.3% and data of different authors showed that the prevalence of this phenomenon in obese individuals varies widely from 6.0-38.4% (35). Evidence existed that lifestyle habits might partly explain the heterogeneity of obesity in terms of metabolic abnormalities. In this study, MHO phenotype occurred more frequently in younger and female twins, and were more likely to exercise and were less likely to smoke or drink. Mechanistically, lifestyle might modulate whole-body energy metabolism and insulin sensitivity. Recently, genome-wide association studies identi ed a set of loci harboring genes possibly controlling both body extra fat distribution (associated with IR) and the metabolic pro le of excess adiposity (i.e., MHO or MUO). This was in according with our within twin-pair analysis. The difference of HOMA-IR was only signi cant between two extreme metabolic phenotypes (0 vs. 4 MetS components) in obese MZ twins, and the difference of HOMA-IR between MHO and MUO was less in MZ twins than DZ twins. These observations suggested a common genetic in uence exist, therefore the difference between the MHO and MUO phenotype may be partly attributed to speci c genetic traits modulating body fat distribution in different regional fat depots which hold diverse biological properties and functions (40).
We found similar hsCRP levels between MHO and MUO group, which differed signi cantly to MHNO, and the association of numbers of MetS components with serum hsCRP began no more signi cant in the model additionally adjusted for obesity indicators. Studies conducted in Mitchelstown cohort participants (41), Wielkopolska general population (42) and a large sample of Brazilian population (22) all reported a similar level of CRP between MHO and MUO group. A study using six sets of criteria to de ne MHO found no signi cant difference of CRP with MUO subjects after multivariate analysis (43). Inconsistent ndings have also been reported that MHO participants had signi cantly lower CRP levels than non-MHO (20,21,44). However, in most studies the difference in CRP levels between MHO and non-MHO subjects became no more statistical signi cance after adjusting for abdominal obesity or percent body fat (43,44) which was in accordance with our nding. These results suggested that abdominal obesity per se is the key role in the progress of subclinical vascular in ammation. Obesity in absence of metabolic risk factors is not entirely benign and MHO population would therefore be expected to have a higher risk of CVD and all-cause mortality than MHNO group (45) The strengths of our study include a twin design, a standard de nition of MetS, adjustment for many available covariates, and various analyses including using strati ed analysis and number of MetS components to test the robustness of the conclusions. However, several potential limitations should be addressed. First, the cross-sectional design can only address associations and not casual relationships. Second, the possibility of residual confounding by unmeasured covariates cannot be excluded. Finally, the gold standard for measurement of insulin resistance is the hyperinsulinemic-euglycemic glucose clamp technique. However, this is a research technique with limited clinical applicability and most studies use HOMA-IR as a clinically useful surrogate measure of insulin resistance.

Conclusion
In conclusion, our ndings demonstrated that metabolic status was independent related to insulin resistance and the highest level was observed in individuals with both obesity and 4 MetS components. Within twin-pair analysis indicated there might exist common genetic in uence between HOMA-IR and MHO/MUO phenotype. Higher in ammation level found among MU subjects might partly be related to abdominal obesity. These ndings provide potential evidence for the different risk assessment based on metabolic status in obese population.

Declarations
Availability of data and materials The data used to support the ndings of this study are available from the corresponding author upon request.