Physiological insulin resistance induced by feeding an HFD for 14 weeks caused a failure of insulin injected into skeletal muscle to be detected in lymph and prevented glucose uptake in response to the injection compared with lean animals. This is reminiscent of previously published results that show experimentally elevated plasma lipids likewise inhibit the ability of injected insulin to diffuse through muscle and initiate glucose uptake (9
). Interestingly, the ability of the HFD to impair insulin dispersion in muscle occurs in the absence of a rise in plasma FFAs, either under basal-fasting conditions or during the clamp.
There was no significant difference between the injected and noninjected arterial insulin in the fat-fed animals, as the arterial supply to both legs should be the same and the insulin injection occurs distal to the arterial sampling point. Also, there is no significant difference between the femoral artery and vein insulin concentrations of the noninjected leg, so the increase observed in the femoral vein insulin concentration of the injected leg was due to insulin overflow from the local area of the injection. Among the high-fat–fed animals, there was variation in the ability of lymph to reflect the more concentrated insulin injections. As the ratio of NINJ lymph to arterial insulin was maintained in the face of increasing systemic insulin, the ability of lymph to detect whole-body insulin changes was not altered.
Lymph measures of interstitial insulin have previously been used in this laboratory (9
) and others (21
), and agree with other measures of interstitial insulin such as microdialysis (25
). Further, gold injected into the interstitium has been shown to concentrate in lymph vessels (26
). Due to the low flow rate and apparent lack of size exclusion of lymph vessels, rapid equilibration of lymph with the intracellular fluid can occur similar to that used in microdialysis, and as such is an appropriate tool for measuring interstitial insulin concentrations, reflecting the makeup of the interstitial fluid to which myocytes are exposed. One limitation in our study is that the lymph vessel insulin concentration is a mixture across the entire leg, whereas insulin is injected into a small area of one muscle; thus the interstitial concentration of insulin in the injected area may be higher than in lymph due to dilution of lymph from uninjected muscle. However, previous results have shown a significant increase in lymph insulin in lean dogs (15
), thus HFD has impaired the appearance of insulin in lymph.
We show that the ability of insulin to access myocytes is impaired in physiological insulin resistance, and several proposed causes of insulin resistance are unable to describe the results we observe here. For example, mitochondrial dysfunction has been proposed as a critical component of insulin resistance (27
), yet in our study, cells are not exposed to elevated insulin, and as such, mitochondrial dysfunction is unlikely to be responsible for the impaired dispersion of insulin. Studies in obese mice have shown that mitochondrial dysfunction is evident in adipose tissue, but not in skeletal muscle (28
), and although stimulating FFA oxidation in skeletal muscle reduces insulin resistance (29
), it does not necessarily follow that mitochondrial function is the primary cause of skeletal muscle insulin resistance. Similarly, it is unlikely that downstream signaling impairment in myocytes (30
) or intracellular fat accumulation (6
) impaired glucose uptake in our study, as there was apparently no measurable elevation of interstitial insulin to cause insulin's cellular effects. However, we cannot discount that myocytes would exhibit insulin resistance if the cells were actually exposed to injected insulin in our model.
The mechanism(s) by which HFD prevents injected insulin from appearing in the interstitial space cannot be clarified from the experiments presented here. As acute hyperlipidemia also resulted in reduced insulin appearance in the interstitial fluid (9
), it is unlikely to be a structural change. One possibility is a lipid-induced alteration in permeability of the endothelial cell (33
), which might allow a more rapid egress of insulin from the interstitial compartment into the bloodstream. However, this permeability theory does not identify the location of the injected insulin prior to appearance in the vein, which may occur as much as 1 h later. It was noted that, although insulin was not detected in lymph, venous insulin had not dropped to preinjection levels even 1 h after injection. As less than 30% of the injected insulin is removed by the vein during the course of the experiment, the remainder must in theory be contained within the muscle, although is not detected in the interstitial space. The excess may be taken up into some cells. Insulin is internalized by endothelial cells en route to the vein (35
), and, as endothelial cells exhibit insulin resistance (36
), this internalization may be increased in obesity to overcome the impaired insulin signaling, thus removing insulin from the interstitial space. As there is no insulin in the interstitial space, only myocytes immediately adjacent to the area of injection may internalize insulin. Alternatively, in lean animals, injected insulin could increase the perfusion of muscle and augment the blood flow to the area, therefore increasing the dispersion of insulin through the muscle (37
). Obese rats (38
) and those with pharmacologically elevated lipids (8
) have impaired insulin-mediated capillary recruitment; insulin injected into the muscle may be trapped in the small area of injection, without the means to increase its dispersion area. It is also possible that the local high insulin concentration after injection is in the interstitial space, although not in an area sampled by the lymph. In lean animals, injected insulin would cause an increase in the distribution volume to allow more complete dissemination of insulin through the muscle, and therefore detection in the lymph. Adiposity would prevent this increase in distribution volume (39
) and reduce the appearance of insulin in the lymph, as less of the muscle is exposed to insulin.
The role of the endothelial cell in the transport of insulin is unclear—evidence exists for both paracellular and transcellular transport of insulin. A recent study has shown that in cell culture, endothelial cells concentrate insulin (38
); whether this occurs in vivo has not been determined, and there is no indication of whether this is unidirectional or can move from plasma to interstitium and back. As lean animals show significant insulin appearance in the vein, insulin can move in both directions. Although endothelial cells exhibit insulin resistance (36
), effects on insulin transport are not known; however, insulin resistance can impair the appearance of insulin in the interstitium (20
), and transport is known to be rate limiting to insulin action (19
Although we have shown that insulin resistance, either chronic (HFD) or acute (9
), prevents dissemination of insulin through the muscle, the mechanism is not yet known. As the HFD does not increase, and in fact suppresses basal plasma FFAs, and isoflurane anesthesia reduces plasma FFAs by 70% (17
), plasma FFAs are unlikely to be the primary cause of the impaired dissemination of injected insulin. However, as plasma lipids are elevated nocturnally by an HFD (13
), it is possible that whereas plasma FFAs are not a direct cause of insulin resistance in our study, tissue FFA levels may be. Intramyocellular lipid content can be altered by diet (11
) and acutely by lipid infusion (40
); as many cells are not exposed to injected insulin, it is unlikely that the intracellular lipid would affect our results, although there is the possibility of extracellular fat accumulation. It is unclear whether lipids must be in the interstitial space to inhibit dispersion of intramuscularly injected insulin, or whether another factor may be inhibiting the dispersion of insulin.
The HFD dogs were subjected to a significantly higher glucose level than the lean animals throughout the injection procedure, as their plasma glucose level after anesthesia and prior to insulin and somatostatin infusion was higher. The arterial glucose of animals infused with lipid is not significantly different from either lean or HFD-fed animals, and values appear to be intermediate (9
). Endothelial dysfunction may be caused by both acute hyperglycemia (41
) and hyperlipidemia (42
), which may prevent insulin-induced capillary recruitment. Thus, impaired insulin dispersion observed with lipid infusion (9
) and fat feeding may result from different plasma factors that ultimately affect the capillary endothelium. Further studies are necessary to determine whether elevated glucose affects the dispersion of injected insulin.
In conclusion, in an HFD, which induces chronic insulin resistance, insulin injected directly into the muscle is not detectable in the interstitial space (lymph), but appears to wash out in the venous blood flow. This is similar to previous results (9
) that demonstrate acute lipid-induced insulin resistance prevented injected insulin from diffusing through the interstitium. The apparent cellular insulin resistance occurs because myocytes are not exposed to insulin; potential downstream effectors such as mitochondrial dysfunction, intramyocellular fat accumulation, and signaling impairment are unlikely to be responsible for the inability of injected insulin to diffuse through the interstitial space in this diet-induced insulin resistance. Although the injection study is not a physiological situation, there are clear differences in how insulin is dealt with in the interstitium, and its access to muscle. Therefore, studies on the insulin sensitivity of individual cells should consider whether insulin was able to access the tissue prior to any perceived defect in insulin signaling or response.