Mitochondrial oxidants, but not respiration, are sensitive to glucose in adipocytes

Abstract

Insulin action in adipose tissue is crucial for whole-body glucose homeostasis, with insulin resistance being a major risk factor for metabolic diseases such as type 2 diabetes. Recent studies have proposed mitochondrial oxidants as a unifying driver of adipose insulin resistance, serving as a signal of nutrient excess. However, neither the substrates for nor sites of oxidant production are known. Since insulin stimulates glucose utilisation, we hypothesised that glucose oxidation would fuel respiration, in turn generating mitochondrial oxidants. This would impair insulin action, limiting further glucose uptake in a negative feedback loop of 'glucose-dependent' insulin resistance. Using primary rat adipocytes and cultured 3T3-L1 adipocytes, we observed that insulin increased respiration, but notably this occurred independently of glucose supply. In contrast, glucose was required for insulin to increase mitochondrial oxidants. Despite rising to similar levels as when treated with other agents that cause insulin resistance, glucosedependent mitochondrial oxidants failed to cause insulin resistance. Subsequent studies revealed a temporal relationship whereby mitochondrial oxidants needed to increase before the insulin stimulus to induce insulin resistance. Together, these data reveal that a) adipocyte respiration is principally fuelled from non-glucose sources, b) there is a disconnect between respiration and oxidative stress, whereby mitochondrial oxidant levels do not rise with increased respiration unless glucose is present, and c) mitochondrial oxidative stress must precede the insulin stimulus to cause insulin resistance, explaining why short-term insulin-dependent glucose utilisation does not promote insulin resistance. These data provide additional clues to mechanistically link nutrient excess to adipose insulin resistance. Adipose tissue is a key nutrient sensor in mammals (1). Being highly sensitive to insulin, adipocytes respond to nutrient replete conditions by increasing energy intake and storage as lipid. Conversely, under nutrient excess, adipocytes become insulin resistant, leading to increased lipid utilisation and reduced glucose intake. Indeed, impaired glucose uptake into adipose tissue is one of the earliest defects observed in whole-body insulin resistance, in both rodents and humans (e.g., (2

Htd2 deficiency-associated suppression of α-lipoic acid production provokes mitochondrial dysfunction and insulin resistance in adipocytes

“…Adipocytes are well acknowledged for actively responding to insulin signal for regulating systemic glucose and lipid homeostasis with the involvement of classic Akt phosphorylation and Glut4 translocation [ 3 ]. Previous studies have highlighted mitochondrial dysfunction as a pivotal factor contributing to disruption of adipose insulin sensitivity [ [52] , [53] , [54] ]. Following observation of mitochondrial oxidative stress induced by Htd2 knockdown, we consistently found that Htd2 knockdown decreased Akt phosphorylation, GLUT4 expression, and glucose uptake under insulin stimuli in both preadipocytes and mature adipocytes, further supporting the direct relation between mitochondrial dysfunction and adipocyte insulin resistance.…”

Insulin signaling requires glucose to promote lipid anabolism in adipocytes

“…In support of this, branched chain amino acids (BCAAs) also contribute substantially as lipogenic substrates in cultured adipocytes, as well as primary adipocytes and adipose tissue from rodents and humans (12)(13)(14)(15). Likewise, we previously observed that glucose is not required for insulin to stimulate respiration in adipocytes (16), which relies on other carbon substrates when glucose is absent. Thus, although anabolic processes such as lipogenesis may serve as a means of glucose storage in adipocytes, it is unclear whether glucose per se is necessary for insulin action.…”

“…1A) and is metabolized more slowly than glucose, such that flux through anabolic pathways becomes dependent on nonglucose sources such as amino acids (19). We previously showed that substituting glucose for galactose in adipocytes abolished the increase in lactate production, but not respiration, in response to insulin (16). We anticipated that if glucose was required for anabolism, flux through anabolic pathways would increase in response to insulin stimulation, but the metabolites in these pathways would become depleted if glucose was substituted with galactose.…”

Kinetic Trans-omic Analysis Reveals Key Regulatory Mechanisms for Insulin-Regulated Glucose Metabolism in Adipocytes

“…Insulin is a key hormone that regulates cellular glucose metabolism. Adipocytes respond to insulin by changing the flux of glucose through various metabolic pathways ( Dimitriadis et al., 2011 ; Giorgino et al., 2005 ; Krycer et al., 2017 , 2020b ; 2020a ; Quek et al., 2020 ). In 14 C-glucose labeling experiments with adipocytes ( Cahill et al., 1959 ; Katz et al., 1966 ), insulin increases fluxes through the metabolic pathways of glucose uptake, glycolysis, oxidative pentose phosphate (PP) pathway, glycogen synthesis, glycerol synthesis, and fatty acid synthesis.…”

“…However, this assumption is physiologically inaccurate particularly in the context of mammalian physiology. For example, in vivo insulin-stimulated changes in the fluxes through metabolic pathways are highly dynamic and are probably never at a steady state ( Krycer et al., 2017 , 2020a ).…”