Effect of circulating ceramides on adiposity and insulin resistance in patients with type 2 diabetes: An observational cross‐sectional study

Abstract Introduction Insulin resistance (IR) is one of the common chronic metabolic disorders in Africa and elsewhere. Accumulation of lipids in the body may be due to an imbalance in the metabolism of lipids, glucose and proteins. Ceramides are a sphingolipid class of lipids that are biologically active and vital in the production of more complex lipids. Circulating ceramides are thought to have a role in the development of obesity‐related IR, although the precise involvement remains unclear. Aim To investigate the impact of circulating ceramide on IR and body adiposity in people with and without type 2 diabetes mellitus (T2DM). Methodology The study was observational and cross‐sectional. There were a total of 84 volunteers with T2DM and 75 nondiabetics (control). The participants' ages, body mass indexes (BMI), waist circumferences, and blood pressure (BP) were among the clinical parameters assessed. Ceramide levels, fasting plasma glucose (FPG), lipids, basal insulin levels and glycated haemoglobin (HbA1c) were also measured. Additionally, the homeostatic model assessment for IR (HOMA‐IR) and beta cell function (HOMA‐β) were computed. Results T2DM and control participants had different mean values for anthropometric parameters, BP, FPG, HbA1c, lipids, insulin, HOMA‐IR, HOMA‐β and ceramide levels (p < .05 for all). HOMA‐IR, HOMA‐β and cardiovascular risk were significant correlates with ceramide levels in the T2DM group (r = 0.24; −0.34; 0.24, p < .05, respectively). Further, FPG (OR = 1.83, p = .01) and ceramide (OR = 1.05, p = .01) levels were significant predictors of IR in the case group. Conclusion Patients with T2DM exhibited high ceramide concentrations, which, when combined with high FPG, were associated with IR. The consequences of circulating ceramides in health and disease; however, merit further research.


| INTRODUC TI ON
Insulin resistance (IR) is characterized by diminished responsiveness of peripheral tissues to the action of insulin; often leading to hyperglycemia. The aforementioned can lead to a reduction in postprandial glucose storage, particularly in the skeletal muscle and liver. [1][2][3] IR is known to affect about 15.5%-46.5% of the human population. 4,5 A sedentary lifestyle, heredity, a high-fat diet, obesity, type 2 diabetes mellitus (T2DM) and heart-related pathologies have all been associated with IR. [6][7][8] Indeed, obesity-mediated IR is common in individuals who live in areas defined by persistent energy surplus, a low basal metabolic rate or a combination of both. [9][10][11] Individuals affected by these metabolic derailments often have vision-related challenges, have their lower limbs amputated and had kidney failure, among others. [12][13][14] There is still a paucity of information as to how obesity can contribute to IR and T2DM. One hypothesis being explored is that when lipids buildup more than their oxidative demands, they spill over into detrimental metabolic pathways. [15][16][17] Ceramides are essential components of bioactive lipids that are produced when sphingomyelin, sphingosine or fatty acids are hydrolysed. [18][19][20] The amount of ceramide that is produced can be controlled by the presence of longchain saturated fatty acids in the endoplasmic reticulum. These fatty acids are involved in the process of de novo ceramide synthesis. 21,22 Ceramide levels have been associated with glucose metabolism dysregulation 23,24 and subsequent IR in humans, albeit the precise mechanism is unknown. Reducing peripheral glucose absorption, inactivating Akt and generating an inflammatory milieu (activating tumour necrosis factor-alpha) are all conceivable mechanisms that have been proposed for how a high concentration of ceramides could cause IR. [25][26][27] In sub-Saharan Africa, there is very little information or data on the role of circulating ceramides and IR. Currently, available treatment options for IR and T2DM focus on lowering cholesterol and triglycerides. However, IR can occur even when cholesterol and triglyceride levels are normal, which suggests that other possible lipids and their intermediates are involved in the process. Knowing basal ceramide levels may have potential implications in the clinical management of patients with metabolic disorders especially in developing countries and in limited-resource settings. The study's goal was to look into the effect of circulatory ceramide on IR and body adiposity in persons with T2DM. We hypothesize that increasing levels of circulating ceramide will be related to an increase in IR and body adiposity among research participants.

| Study design, site and participants
This was an observational cross-sectional research. One hundred and fifty-nine (159) volunteers were recruited from the St. Gregory Catholic Hospital (SGCH), which is located in Gomoa Buduburam, Central Region, Ghana. Of the participants, 84 were diagnosed with T2DM, while 75 did not have DM, and served as control. Participants were to be 18 years and above. The control group had no history of T2DM and had fasting plasma glucose (FPG) levels that were lower than 5.6 mmol/L. Further, our control group had glycated haemoglobin (HbA1c) levels between 4% and 5.6%, and thus met the criteria in the classification of a nondiabetic. 28  . Before recruitment, participants were required to complete an informed consent form indicating that they were willing to take part in the study.
The participants were given information on the following when they signed their consent forms: the rationale and duration of the study, the benefit of the study, the materials used in sample collection and the possible dangers of the sampling procedure. It was requested of the participants that they comply with all COVID-19 protocols. A T2DM prevalence rate of 6.46% was used in the calculation of the sample size. 29

| Clinical assessment and laboratory procedures
To obtain sociodemographic and clinical information from research participants, a standard questionnaire was employed in conjunction with a review of patients' folders. The questionnaire obtained information about the participants' names, ages, gender, marital status, living styles, family history of DM and times of onset of DM.
The weights of the participants were measured by using a weighing scale (Omron, USA). A wall-mounted stadiometer was used to measure the height of subjects. Body weight and height measured were to 1.0 kg and 0.005 m of their approximate measurements, respectively. Participants wore little clothing and were barefoot when their heights and weights were measured. Body mass index (BMI) was determined by dividing weight by the square of height (Kg/m 2 ).
BMI was further used to categorize patients into obese 1 (30-34.9 Kg/m 2 ), obese 2 (35-39.9 Kg/m 2 ) and obese 3 (≥40 Kg/m 2 ). 30 Using a tape measure, standing waist circumference (WC) to the nearest 0.1 cm around the navel or at the level of the umbilicus was measured. In addition, blood pressure (BP) was recorded after 10-15 min of rest while the volunteer sat in a chair. The BP was measured using an automated cuff BP machine. A validated formula was used to estimate the body fat percentage. For men, the formula was 0.567 × WC + 0.101 × age (yrs.) − 31.8, while for females, the formula was 0.438 × WC + 0.221 × age (yrs.) − 9.4. 31 Before blood sample collection, the participants were instructed to abstain from food for 10-14 h overnight. A normal blood draw of 5 mL was performed on each participant while they were fasting by a qualified phlebotomist during the hours of 7:00-9:00 AM.
Quantification of glycosylated haemoglobin (HbA1c) and fasting plasma glucose (FBP) was performed using 1 mL of the blood sample that was in an EDTA tube and another 1 mL of blood that was in a fluoride oxalate tube, respectively. To obtain sera, the remaining 3 mL were transferred to a serum separator tube, which was then put on a rack at a temperature of 25°C for 10-20 min before being centrifuged at 3000 rpm for 10 min. The resulting sera were divided into 0.5 mL portions and placed in Eppendorf tubes. These were then kept at a temperature of −20°C for about a month until analysis. The sandwich ELISA approach (GenWay Biotech Inc) was used to determine the ceramide and insulin concentrations present in sera.
The test involved the engagement of a double-specific monoclonal antibody to provide accurate results. Fluorescence immunoassay (ichorma™) was used to determine the HbA1c levels in the blood (Boditech Med Inc). An automated chemical analyser was used to obtain concentrations of FPG and lipids (Mindray BS430, China).
The homeostatic assessment for IR (HOMA-IR) and beta cell function (HOMAβ) were computed employing a formula that has been previously described. 32,33

| RE SULTS
The study had a total of 149 participants, including 84 with T2DM (the case group) and 75 as controls. In the case group, there were 68 females, making up 81% of the total. In the control group, there were 47 females, making up 62.7% of the total. The majority (42%) of the cases had completed basic education in contrast to the control group where almost all (90.7%) had completed tertiary education.
Moreover, about a third (21.4%) in the case group were insulinsensitive ( Table 1).
The clinical (continuous), anthropometric and biochemical information of the study population is displayed in Table 2. The average age of the participants in the case group was 53.7 ± 9.5 years, whereas the average age of the volunteers in the control group was 31.9 ± 6.5 years. The case group had higher WC, waist-to-hip ratios, BMI, percentages of body fat, blood pressure (both systolic and diastolic), ceramide levels, FPG, total cholesterol, triglycerides, LDL-cholesterol and HbA1c levels than the control group (p < .05 for all). On the contrary, insulin levels and HOMAβ were higher in the control group compared with the case group (p < .05 for both).
HOMA-IR levels were, however, comparable between the cases and controls (2.91 ± 1.35 vs. 2.55 ± 1.54; p = .092). Table 3  and serum ceramides (odds ratio = 1.05, p = .01) were found to be significant predictors of IR among the case-cohort (about two and one-fold higher potential predictability risks, respectively) (

| DISCUSS ION
The primary objective of this study was to determine whether or not individuals in Ghana with T2DM who were obese had altered levels of circulatory ceramides. It is well known that the capacity of sphingolipids to regulate cellular activity is of importance in several bodily processes (physiological and pathological). In this study, participants with TDM had higher serum ceramide levels than their nondiabetic counterparts. Broskey and colleagues earlier reported that subjects who were both obese and diabetic had higher levels of ceramides in their skeletal muscle, while subjects who were neither obese nor diabetic had significantly lower levels of ceramides in their skeletal muscle. 34 Indeed, the plasma concentration of dihydroceramide, a precursor to ceramide, has been linked to an increased likelihood of developing diabetes in mice and humans. 35 Within our case population, the metrics of insulin resistance, insulin sensitivity and cardiovascular risk all demonstrated significant relationships with ceramide levels and agreed with prior studies 36 (Table 3). In both human and animal models, ceramide levels in the blood, adipose tissue, the liver and skeletal muscle are inversely correlated with the degree of insulin sensitivity. [36][37][38] Although not observed in the present study, a recent study lends support to the observation that ceramide accumulation plays a role in sarcopenia that is detrimental to the body. 39 The authors concluded that as people get older, ceramides begin to buildup in the muscle, hence affecting the function of the muscle. Yet, older persons who had a genetic mechanism that reduced ceramide production in their muscle tissues were fitter than expected for their age, evidenced by greater handgrip strength, as well as the capacity to walk long distances and stand up from a chair. Thus, ceramides in muscle tissues and other parts of the body may be the mediators of multiple processes in cells. Ageing skeletal muscle has been associated with greater muscle ceramides in some studies 40 to the menopausal status of the participants, it was discovered that the levels of long-chain ceramides, very long-chain ceramides and dihydroceramide were higher in postmenopausal women than they were in premenopausal women. The same study found a substantial inverse association between the circulating levels of estradiol and ceramide (d18:1/24:1) in women of all ages. This correlation was not found in men, which suggests that estradiol may regulate the circulating levels of ceramide in a sexually dimorphic manner.
Ceramides have also been reported to vary with oestrogen levels. In their study of older female adults, Kendall and colleagues recently discovered a correlation between advancing age, ele- however, these reductions were not observed in postmenopausal women who were using hormone replacement therapy (HRT). In addition, the treatment of primary human keratinocytes with estradiol (10 nM) led to an increase in the formation of ceramides, which suggests that oestrogen plays a direct regulatory role in the metabolism of keratinocyte ceramides. 45 Another in vitro study conducted with human cancer cells that expressed oestrogen receptors showed that a considerable reduction in ceramide buildup could be achieved through incubation with estradiol. 44 Taken together, these findings show that ageing is linked to levels of circulating ceramide, which, in postmenopausal women, appear to be regulated by levels of estradiol.
Additionally, BMI, waist-to-height ratio, insulin resistance, adiposity, percent body fat, waist circumference and waist-to-hip ratio were all significant correlates in the control group and were consistent with those found in previous research. 46  The deleterious effects of ceramides have also been shown in muscles during Duchenne disease. 55 Insulin sensitivity (HOMAβ) was found to be associated with a reduction in total ceramide concentration and agreed with the findings of a prior study denoted as the strong heart family study (SHFS). The study discovered various ceramide species that were related to fasting insulin levels, HOMA-IR levels and HOMA-B levels both at the baseline and follow-up. 56  Abbreviations: AIP, an atherogenic index of plasma; BMI, body mass index; BP, blood pressure; FPG, fasting plasma glucose; HbA1c, glycated haemoglobin; HDL-C, high-density lipoprotein cholesterol; HOMA-IR, the homeostatic model assessment of insulin resistance; HOMAβ, the homeostatic model assessment of beta cell function; LDL-C, low-density lipoprotein cholesterol.
In this table shows the relationship between levels of ceramide and several clinical and biochemical correlates of the study participants. r is the correlation coefficient. p-value was considered significant at .05 alpha level.
subjects. The levels of ceramide subspecies, as well as selected biochemical parameters, were determined before surgery, three Ceramides promote LDL entry into vessel walls, which in turn encourages plaque development. The existence of a positive connection between LDL-ceramide and insulin resistance (HOMA-IR) lends weight to the notion that LDL-ceramide plays a pathogenic role. In a separate but related study, circulating LDL-ceramides led to IR and inflammation in lean mice, and they decreased glucose disposal uptake in cultured myotubes. 37  Although a correlation between TG/HDL ratio and IR has been well documented in the context of major clinical studies, the present investigation could not find a meaningful connection between the two. For example, a higher triglyceride to high-density lipoprotein cholesterol ratio (TG/HDL) was found to be an independent predictive factor and positively linked with an increased risk of incident diabetes in a recent large retrospective study that was conducted on Chinese males who were at least 20 years old. 64 Another large cross-sectional study, with 1608 nonobese middle-aged and elderly individuals, found that the TG/HDL-C ratio was the best marker for predicting IR in the nonobese subjects, with the area under the receiver operating characteristic curve (AUC) of 0.73, p < .001. Increased TG/HDL-C ratio was related to IR and cell function in Hispanic and African American men and women, similarly to Caucasians, and could predict the incidence of type 2 diabetes in women. 65 It would appear that this is the first time that the levels of cera-

PA RTI CI PA NT CO N S ENT S TATEM ENT
All participants provided written informed consent.

CLI N I C A L TR I A L R EG I S TR ATI O N
The study protocol has not been registered as a clinical trial. writing -review and editing (supporting).

ACK N O WLE D G E M ENTS
The authors wish to thank the management and staff of both St.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no competing financial interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The corresponding author will provide the interested party with access to the datasets that were used throughout this study upon reasonable request.

E TH I C A L A PPROVA L S TATE M E NT
The