PLoS ONE 2014; September 9(9): e107140

Reduction of Specific Circulating Lymphocyte Populations with Metabolic Risk Factors in Patients at Risk to Develop Type 2 Diabetes.

Helena Cucak1, Dorte Vistisen2, Daniel Witte2, Annelotte Philipsen2, Alexander Rosendahl1, 3*


1 Hagedorn Research Institute. Department of Diabetic Complications Biology Novo Nordisk A/S, Maløv, Denmark

2 Department of Clinical Epidemiology, Steno Diabetes Center, Gentofte, Denmark

3 Department of New Heamophilia, Novo Nordisk A/S, Gentofte, Denmark

* Corresponding author



Low-grade inflammation, characterized by increased pro-inflammatory cytokine levels, is present in patients with obesity linked insulin resistance, hyperglycemia and hyperlipidemia and considered to play a leading role to progression into type 2 diabetes (T2D). In adipose tissue in obese patients and in pancreatic islets in T2D patients cellular inflammation is present. However, the systemic leukocyte compartment and the circulating endothelial/precursor compartment in patients at risk to develop T2D has so far not been analyzed in detail. To address this, peripheral blood cells from a cohort of 20 subjects at risk to develop diabetes with normal to impaired glucose tolerance were analyzed by flow cytometry using a wide range of cellular markers and correlated to known metabolic risk factors for T2D i.e. fasting plasma glucose (FPG), 2 h plasma glucose (2 h PG), HbA1c, body mass index (BMI), homeostasis model assessment of b-cell function (HOMA-B), homeostasis model assessment of insulin sensitivity (HOMA-IS) and fasting insulin (FI). The four highest ranked cell markers for each risk factor were identified by random forest analysis. In the cohort, a significant negative correlation between the number of TLR4+ CD4 T cells and increased FPG was demonstrated. Similarly, with increased BMI the frequency of TLR4+ B cells was significantly decreased, as was the frequency of IL-21R+ CD4 T cells. Unlinked to metabolic risk factors, the frequency of regulatory T cells was reduced and TLR4+ CD4 T cells were increased with age. Taken together, in this small cohort of subjects at risk to develop T2D, a modulation of the circulating immune cell pool was demonstrated to correlate with risk factors like FPG and BMI. This may provide novel insights into the inflammatory mechanisms involved in the progression to diabetes in subjects at risk.

PMID: 25254631




During the last decades an epidemic increase of overweight and obesity has been observed particularly in industrial countries where close to 20% of the adult population now is considered obese with a BMI above 30 [1]. Type 2 diabetes (T2D) is showing a parallel increase pattern and several studies has demonstrated direct linkage from overweight to obesity leading to metabolic disease like T2D [2,3]. If the number of T2D subjects continue to increase in a similar rate as now it is estimated that close to 0.5 billion people around the globe will have established T2D by 2030 [4]. This will put the health care system under substantial stress when considering that T2D patients have a significantly enhanced risk to develop diabetes associated complications like diabetic nephropathy, stroke and macro vascular diseases like atherosclerosis [5,6]. Therefore early identification of potential future T2D patients before clinical signs are established will provide a great benefit to society.

Recently significant progress has been made showing a close link between the inflammation and metabolic diseases [7,8]. In fact, the so called low grade inflammation present in adipose tissue and skeletal muscle in obese patients and animal models is required for development of insulin resistance [9]. Patients suffering from metabolic disease have an increased number of white blood cells [10]. These cells, in response to chemokines in the blood, migrate specifically into tissues where they are further activated and polarized into effector cells (8). Recently, T2D was hypothesized to carry characteristics of an inflammatory disease as well [7]. Patients have elevated levels of acute-phase proteins and cytokines in their blood that are modulated during disease progression [11]. In fact, the cells undergo maturation during the disease from early pro-inflammatory cells into chronic tissue remodeling cells that may participate in complications like diabetic nephropathy (12,13).

We hypnotized that if we could identify a specific leukocyte or stem cell population in the blood from patients at risk to develop T2D, these patients could be identified earlier and hence receive appropriate treatment and risk progressing into T2D. As food is known modulate white blood composition, we performed analysis to determine if food intake in these patients was linked to risk factors for disease development.

The vast majority of the studies evaluating inflammation in patients with obesity have focused on monocytes and macrophages in tissues such as adipose tissue. However, to identify an easily accessible biomarker, evaluation in blood is a superior. Therefore, we conducted a detailed analysis of the blood before and after food intake. As it turns out, neither the frequency of monocytes nor the so-called polarized M1/M2-like monocytes were altered by food intake. Likewise, the effector monocytes recognizing bacterial pathogens and cytokine responsive cells were not affected by food intake.



AR fig1

Figure 1. Frequency of monocytes and monocyte subsets are not modulated in patients at risk to develop diabetes after food intake. Total frequency of CD68+ monocytes pre and post food intake is not changed (A), nor is the frequency of M1-like (CD68+CD1632/intCD11chigh) or the frequency of M2-like (CD68+CD163int/hiCD11cint) cells (B) or the frequency of innate TLR4 expressing or cytokine responsive IL-21R positive monocytes (C).


Although substantial evidence is present for T cells in T1D disease, T2D is not considered to involve lymphocytes and hence the blood lymphocyte count was not anticipated to be modulated in our cohort. In line with the expectation, we did not observe any modulation of the large CD4 and CD8 T cell normally detected in differential counts at hospitals. To our great surprise, we demonstrated that a very small T cell subpopulation expressing the TLR4 receptor was significantly increased in our patients after food intake. This population has been well described to participate in regulatory functions controlling specific parts of the inflammatory responses. In fact, when we performed statistical evaluation of our data we demonstrated that the TLR4 expressing T cells were negatively correlated with increase of fasting plasma glucose levels before intake of food. In addition, when we further evaluated blood phenotypes we could demonstrate a highly significant reduction of IL-21 cytokine responsive effector cells with increasing BMI. This suggests that the more obese a subject is the less IL-21 cytokine responsive effector T cells are present in the blood. Finally, we also demonstrated that the number of regulatory TLR4+ B cells decreased with obesity.


 AR fig2

Figure 2. Regulatory T lymphocytes are associated with risk factors to develop diabetes. A marked upregulation of TLR4+ T lymphocytes in peripheral blood after food intake (A, B). Number of TLR4+ T cell are negatively associated with fasting plasma glucose (C), Frequency of TLR4+ B cells are negatively associated with BMI (D) and number of IL-21R T cells are negatively associated with BMI (E).


Importance of this study is two-fold. First we demonstrate that even in a small cohort of patients at risk to develop diabetes significant changes in circulating blood leukocyte populations can be detected providing useful information as novel diagnostic biomarkers for early detection of patients likely to progress into disease. These cells populations include small lymphocyte subsets belonging to the regulatory subset of T cells and demonstrate that these regulatory lymphocytes are inversely correlated to an increase of fasting plasma glucose and BMI. Secondly, we demonstrate that intake of food significantly influence the leukocyte subsets in blood. This demonstrates the need for adjustment for food intake when evaluating circulating biomarkers in blood.



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Corresponding author: 

Alexander Rosendahl PhD.

Hagedorn Research Institute and Department of Diabetic Complications and New Heamophilia, Novo Nordisk A/S

Novo Nordisk Park, 2760 Måløv, Denmark


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