Lipid handling in models of insulin resistance and physical (in)activity

Worldwide, diabetes has reached epidemic proportions, with type 2 diabetes accounting for ~90% of all cases. One of the earliest disturbances in the etiology of type 2 diabetes is insulin resistance in major metabolic tissues, such as the liver and skeletal muscle. It has been well established that high-energy, high-fat diets1-3 and a sedentary lifestyle4-7 are important factors leading to an increased risk of developing type 2 diabetes. Whole-body insulin resistance is generally accompanied by ectopic lipid deposition in liver8, 9 and skeletal muscle tissue.10-12 The mechanistic link between intracellular lipid overload and insulin resistance is believed to reside in the accumulation of lipid derived intermediates, such as diacylglycerols and ceramides, which trigger activation of novel protein kinases C leading to impairments in insulin signaling.13 A key issue that remains to be determined is whether the excess storage of lipids in insulin-resistant muscle and liver is a consequence of greater lipid uptake from the circulation, decreased lipid utilization, or a combination of both. To identify the exact origin of lipid handling derangements in insulinresistant tissues, direct in vivo measurements of lipid storage dynamics are essential. Lipid handling in insulin-resistant liver and skeletal muscle has previously been determined using radioactive or stable-isotope labeled fatty acid tracers in arterio-venous balance methods or by determining the specific uptake of these tracers in biopsies, collected tissues, cultured liver and muscle cells, or giant vesicles obtained from muscle. However, these methods are either indirect, invasive, or only performed in vitro. Localized 1H magnetic resonance spectroscopy (MRS) is a powerful tool for the noninvasive detection of intracellular lipid storage in vivo, but it cannot discriminate between disturbances in lipid uptake on one hand and lipid utilization on the other. In chapter 2, the novel application of localized 1H-[13C] MRS in combination with the oral administration of a 13C-labeled lipid mixture was introduced to examine in vivo lipid handling in liver and skeletal muscle in rodents. 1H-[13C] MRS indirectly detects the 13C-labeled lipids through the attached 1H nuclei, which has a number of advantages over the direct detection of 13C: (i) it is more sensitive than direct 13C MRS;14-16 (ii) it can be combined with 1H single-voxel localization, which is less prone to chemical shift displacement errors compared with 13C volume selection;17 (iii) 1H MRS separately detects intra- and extracellular lipids based on a difference in resonance frequency,18-20 which is not apparent in the 13C MR spectra; and (iv) in contrast to direct 13C MRS, no external reference is needed for the quantification of the amount of 13Clabeled lipids from the 1H-[13C] MR spectrum, because it can be expressed relative to the total (12C+13C) amount of lipids or the water signal, which are both determined within the same experiment. 1H-[13C] MRS proved to be a powerful method for the longitudinal assessment of in vivo postprandial lipid partitioning in liver and skeletal muscle of healthy rats. We have shown that, in healthy rats, the uptake of 13C-labeled lipids was about 10-fold higher in liver compared with skeletal muscle and that between 5 and 24 h after 13C-labeled lipid administration the turnover of 13C-enriched lipids was more rapid in liver than in skeletal muscle tissue. Results from studies on intracellular lipid handling in insulinresistant skeletal muscle and liver tissue are far from consistent. Lipid uptake in muscle of insulin-resistant and type 2 diabetic patients as well as animal models has been reported to be increased, similar or even decreased when compared with insulin-sensitive controls. Also for the proposed differences in muscle lipid oxidation and liver lipid uptake and secretion between healthy and insulin-resistant and/or type 2 diabetic conditions inconsistent data have been reported. Differences in study design, such as the nutritional state (postprandial versus postabsorptive conditions), the stage of type 2 diabetes pathogenesis and the methodology applied to assess lipid handling, likely contributed to the apparent inconsistencies. In chapter 3, the 1H-[13C] MRS technique was applied in pre-diabetic and diabetic rats to gain more insight in the derangements in liver and skeletal muscle lipid handling at different stages during the pathogenesis of type 2 diabetes. It was shown that in vivo postprandial lipid handling was disturbed in both liver and skeletal muscle of pre-diabetic and diabetic rats compared with healthy controls. However, important differences appeared to exist between the pre-diabetic and diabetic state. Lipid uptake in liver was largely elevated in the pre-diabetic state, whereas muscle seemed to be protected from excess lipid uptake. In contrast, after the development of overt type 2 diabetes, lipid uptake was strongly elevated in both liver and muscle. Muscle lipid utilization was significantly lower in both pre-diabetic and diabetic muscle, indicative of impairments in lipid mobilization and/or oxidation. In chapter 3, it was postulated that the accumulation of lipids in pre-diabetic muscle is mainly the result of reduced lipid oxidation, but that the situation is aggravated during the development to overt diabetes due to an additional increased uptake of dietary lipids. Besides the consumption of high-energy, high-fat diets,1-3 the increased incidence of type 2 diabetes, in particular in children and adolescents, is also attributed to the sedentary lifestyles4-7 adapted in developed countries.21, 22 Previous studies showed that individuals who are largely dependent on a wheelchair for mobility have an increased risk of developing glucose intolerance and insulin resistance,23-26 which is likely attributed to reduced physical activity and loss of leg muscle mass. In chapter 4, the impact of chronic muscle deconditioning on whole-body insulin sensitivity, muscle oxidative capacity and intramyocellular lipid (IMCL) content was assessed in subjects with paraplegia. IMCL content was determined both in vivo and in vitro using 1H MRS and fluorescence microscopy, respectively. Moreover, muscle biopsy samples were stained for succinate-dehydrogenase (SDH) activity as a measure for muscle fiber oxidative capacity. Despite the loss of functional ability of the legs, young, healthy paraplegic individuals did not show a substantial decline in glucose tolerance and/or whole-body insulin sensitivity. The loss of leg muscle recruitment was accompanied by lower muscle fiber oxidative capacity, whereas mixed muscle lipid deposition remained unaffected. However, the typical subcellular SDH and IMCL distribution patterns, as observed in able-bodied controls, were lost in muscle fibers collected from subjects with paraplegia. These findings suggest that in healthy subjects negative feedback systems prevent excess lipid deposition in muscle tissue during an extensive period of muscle disuse, reflecting an adaptive response to a lower metabolic demand. In contrast to the negative effects of a sedentary lifestyle, physical activity has been shown to be beneficial in the treatment of obesity-related disorders, such as insulin resistance and type 2 diabetes.27 It is well accepted that both endurance and resistance exercise training increase whole-body glucose tolerance and/or insulin sensitivity,28, 29 but the effects of exercise on lipid handling in insulin-resistant muscle and, in particular, liver are not well understood. In chapter 5, the effect of a single bout of exercise on postprandial lipid handling in liver and skeletal muscle was investigated, both in healthy and in type 2 diabetic rats. Directly after a single bout of endurance exercise, 13C-labeled lipids were orally administered to the rats and 1H-[13C] MRS was applied to examine in vivo lipid partitioning. One hour of treadmill running depleted the IMCL pool, but did not affect total liver lipid content in both non-diabetic and diabetic rats. Although the total IMCL pool was replenished at 24 h post exercise, prior exercise did not augment postprandial lipid uptake in muscle or liver tissue. Therefore, this study suggests that after exercise, endogenous lipid pools are utilized for the replenishment of IMCL stores, in favor of newly ingested lipids