Hyperinsulinemia is associated with adipocyte hypertrophy and mitochondrial dysfunction

Abstract

Insulin resistance is often associated with hypertrophy of adipocytes. Furthermore, hyperinsulinaemia may mediatechanges in cellular mitochondrial biogenesis and function. Therefore, the current study investigated the hypothesis that lower mitochondrial mass and mitochondrial uncoupling (whereby the electron transport is not used to drive ATP synthesis) is a feature of hypertrophied adipocytes.Methods. Adipose tissue (sub-cutaneous and omental) and blood was obtained from morbidly obese patients (BMI ≥ 40 kg/m2, age 29 ± 2.8 years), undergoing bariatric weight reduction surgery. Anthropometric data was recorded. The blood was used for systemic determination of glucose and insulin by commercial methods. The adipose tissue was separated into the adipocyte and stromal vascular fractions by collagenase digestion. Paraffinembedded adipose tissue was stained with Hematoxylin and Eosin and Image J software was used to determine cell size.Total RNA was isolated using Tri-reagent and the transcriptomeanalyzed using Affymetrix whole transcriptome arrays. The ArrayAnalysis.org statistics module using the Limma package of R/Bioconductor was used for the statistical comparison between High insulin subjects vs. Low insulin group. Genes were considered to be differentially expressed when their absolute log2 fold change (FC) > = 1, and p-value < = 0.05.Indices of mitochondrial function was examined in an in vitro model using 3T3-F442A murine differentiated adipocytes.Cellular lipid content and mitochondrial membrane potential(DΨm) were determined by confocal microscopy with 40 μMBodipy 493/503, a neutral lipid dye, and TMRE (30 nM).NADH/flavoprotein autofluorescence was measured in ‘multitracking’ mode, in which fluorescence was excitedalternately at 351 nm (signal measured at 435-475 nm; NADH)and at 458 (emission measured at >505 nm; flavoprotein). Theresting level of each coenzyme was expressed as a function ofthe maximally oxidized (with the uncoupler FCCP, 1 μM) andmaximally reduced signal (with cyanide, 1 mM). Oxygenconsumption was measured using a Clarke electrode and ATP generation using a luciferase assay.Results.The patient population was dichotomized into hyperinsulinaemic(>7.0 μU/ml) and normoinsulinaemic ( < 6.5 μU/ml) groups. The groups were matched for age and BMI. The hyperinsulianemicgroup, compared to those with normoinsulinaemia, had significantly greater numbers of hypertrophied adipocytes (p = 0.04). Pathway analysis showed that genes involved in the mitochondrial biogenesis (e.g: NRF1, GABPA, TFAM, POLRMT, MTERF and SP1) were comparable between the two groups. However several genes related the electron transport chain and ATP synthesis were significantly different between the hyperinsulinaemic and normoinsulinaemic groups (See table below). Expression of genes related to the electron transport and ATP synthesisComplex ? NDUFA4, ND3,ND6Complex IISDHA, SDHB, SDHC. Complex IIIUQCRH, UQCRFS1.Complex IVCOX5A, COX6A, COX7A. Complex VATP5A, ATP5E, ATP5HAdenine Nucleotide TranslocaterSLC25A4 In adipocytes characterized as having >40% intracellular lipid (hypertrophied), compared to those with < 20% (normal adipocyte), lipid, the rate of oxygen consumption increased(380%). DΨm was significantly reduced in cells with high,compared to those with low, intracellular lipid. Assessment ofredox state in lipid-engorged cells showed that flavoproteinswere more oxidized than NADH, consistent with a shift to b-oxidation as a dominant metabolic pathway. Resting levels ofboth coenzymes were significantly oxidized in lipid-engorgedcells compared to those with < span class = »bumpedFont15