Liver Fat Content Linked to SC Adipose Tissue in T2DM
Obese patients with T2DM who were on stable insulin treatment were included in the study. Exclusion criteria were other types of diabetes, significant cardiovascular, renal, liver or other co-morbidity, use of corticosteroids, uncontrolled endocrine disorders (stable supplementation with thyroid hormone was allowed), bariatric treatment, excessive alcohol consumption (>20 g/day), drug abuse and use of thiazolidinedione derivatives.
Patient demographics, medication, and insulin type and dose were recorded. Body weight, height, waist- and hip-circumference and blood pressure were measured using standard procedures.
Fasting blood samples were drawn to determine glycated haemoglobin (HbA1c), lipids, and free fatty acids (FFAs) (Cobas Mira Plus®; Roche Diagnostics Ltd., Basal, Switzerland).
Total-body DEXA scanning (Hologic QDR 4500 densitometer, Bedford, MA, USA) was used to determine total fat mass and trunk fat. Magnetic resonance (MR) measurements were performed on a Tim-Trio MR system (Siemens, Erlangen, Germany). A series of T1-weighted (flash 2D) axial MR images was acquired from a region extending from 4 cm above to 4 cm below the fourth to fifth lumbar interspace. Visceral and subcutaneous fat areas were determined based on signal intensity. Liver fat was assessed by proton MR spectra (MRS) as described previously.
Subcutaneous (sc) adipose tissue biopsies were obtained under local anaesthesia by needle biopsies performed 6–10 cm lateral to the umbilicus, after an overnight fast. Morphometry of individual fat cells was assessed using digital image analyses as described previously. For each subject, the adipocyte cell diameter of all fat cells in five to ten microscopic fields of view were counted and measured. On average, ~700 fat cells were measured per specimen (range: 150–1500). Adipocyte cell size distribution was expressed as ratio small (≤50 μm):large adipocytes (≥100 μm). For detection of macrophages, adipose tissue sections were incubated with a CD68-monoclonal antibody (AbD Serotec, Oxford, UK). The percentage of macrophages was expressed as the total number of CD68-positive cells divided by the total number of adipocytes counted in 20 random microscopic fields of view ×100. A crown-like structure was defined as an adipocyte surrounded by at least three macrophages.
Protein levels within the adipose tissue were measured by Luminex fluorescent bead human cytokine immunoassays (MILLIPLEX MAP, Millipore Corp., Billerica, MA, USA). Briefly, adipose tissue lysates were prepared using the milliplex map lysis buffer (Millipore) and protein concentrations of Interleukin-1 beta (IL-1b), Interleukin-6 and 8 (IL-6/8), tumour-necrosis factor alfa (TNF-α), leptin, adiponectin, monocyte chemoattractant protein (MCP)-1, resistin and plasminogen activator inhibitor-1 (PAI-1) were determined. Equal amounts of protein were analysed using a Bioplex system (Biorad, Hercules, CA, USA).
The ethical committee of the Radboud University Nijmegen Medical Centre approved the study protocol. All subjects provided written informed consent.
Variables are expressed as means ± SEM. Correlations were calculated by Spearman rank correlation analyses, unless otherwise specified. All calculations were performed using spss software (version 16·0; SPSS Inc., Chicago, IL, USA). Two-tailed P < 0·05 was considered significant.
Patients and Methods
Study Population
Obese patients with T2DM who were on stable insulin treatment were included in the study. Exclusion criteria were other types of diabetes, significant cardiovascular, renal, liver or other co-morbidity, use of corticosteroids, uncontrolled endocrine disorders (stable supplementation with thyroid hormone was allowed), bariatric treatment, excessive alcohol consumption (>20 g/day), drug abuse and use of thiazolidinedione derivatives.
Demographic and Clinical Characteristics
Patient demographics, medication, and insulin type and dose were recorded. Body weight, height, waist- and hip-circumference and blood pressure were measured using standard procedures.
Biochemical Analyses
Fasting blood samples were drawn to determine glycated haemoglobin (HbA1c), lipids, and free fatty acids (FFAs) (Cobas Mira Plus®; Roche Diagnostics Ltd., Basal, Switzerland).
Body Composition and Liver Fat Content
Total-body DEXA scanning (Hologic QDR 4500 densitometer, Bedford, MA, USA) was used to determine total fat mass and trunk fat. Magnetic resonance (MR) measurements were performed on a Tim-Trio MR system (Siemens, Erlangen, Germany). A series of T1-weighted (flash 2D) axial MR images was acquired from a region extending from 4 cm above to 4 cm below the fourth to fifth lumbar interspace. Visceral and subcutaneous fat areas were determined based on signal intensity. Liver fat was assessed by proton MR spectra (MRS) as described previously.
Subcutaneous Adipose Tissue Biopsies
Subcutaneous (sc) adipose tissue biopsies were obtained under local anaesthesia by needle biopsies performed 6–10 cm lateral to the umbilicus, after an overnight fast. Morphometry of individual fat cells was assessed using digital image analyses as described previously. For each subject, the adipocyte cell diameter of all fat cells in five to ten microscopic fields of view were counted and measured. On average, ~700 fat cells were measured per specimen (range: 150–1500). Adipocyte cell size distribution was expressed as ratio small (≤50 μm):large adipocytes (≥100 μm). For detection of macrophages, adipose tissue sections were incubated with a CD68-monoclonal antibody (AbD Serotec, Oxford, UK). The percentage of macrophages was expressed as the total number of CD68-positive cells divided by the total number of adipocytes counted in 20 random microscopic fields of view ×100. A crown-like structure was defined as an adipocyte surrounded by at least three macrophages.
Protein levels within the adipose tissue were measured by Luminex fluorescent bead human cytokine immunoassays (MILLIPLEX MAP, Millipore Corp., Billerica, MA, USA). Briefly, adipose tissue lysates were prepared using the milliplex map lysis buffer (Millipore) and protein concentrations of Interleukin-1 beta (IL-1b), Interleukin-6 and 8 (IL-6/8), tumour-necrosis factor alfa (TNF-α), leptin, adiponectin, monocyte chemoattractant protein (MCP)-1, resistin and plasminogen activator inhibitor-1 (PAI-1) were determined. Equal amounts of protein were analysed using a Bioplex system (Biorad, Hercules, CA, USA).
Informed Consent
The ethical committee of the Radboud University Nijmegen Medical Centre approved the study protocol. All subjects provided written informed consent.
Statistical Analyses
Variables are expressed as means ± SEM. Correlations were calculated by Spearman rank correlation analyses, unless otherwise specified. All calculations were performed using spss software (version 16·0; SPSS Inc., Chicago, IL, USA). Two-tailed P < 0·05 was considered significant.
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