Macronutrient intake, insulin secretion, oxidative stress & inflammation: Clinico-pathological implications.

The landmark observation that adipose tissue in the insulin-resistant ob/ob mouse expresses an excess of the proinflammatory cytokine, tumour necrosis factor-alpha (TNF-α) and that the infusion of soluble TNF-α receptor reverses the insulin-resistant state through neutralization of TNF-α was a conceptual leap and created a veritable intellectual explosion1. This was followed by the demonstration that obese humans not only had elevated concentrations of TNF-α which fell after dietary restriction and weight loss2 but also had increased levels of oxidative stress which also fell rapidly after caloric restriction and weight loss3. It was further shown that TNF-α interfered with insulin signal transduction at the insulin receptor substrate-1 (IRS-1) level4. Further exploration revealed that several serine kinases induced by inflammatory mediators caused serine phosphorylation of IRS-1 to interfere with insulin signaling5. These included protein kinase C-β (PKC-β), mammalian target of rapamycin (m-TOR), S-6-K, c-Jun N-terminal kinase-1 (JNK-1) and p38 mitogen-activated protein kinase (p38MAPK). In addition, suppressor of cytokine signalling-3 (SOCS-3) causes ubiquitylation and proteolysis of IRS-16 while protein-tyrosine phosphatase 1B (PTP-1B) dephosphorylates tyrosine residues on the beta subunit of the insulin receptor7. Thus it became clear that obesity was associated with insulin resistance through inflammatory mechanisms8.

The question that arises, therefore, is what induces chronic inflammation in the obese? The answer lies in the simple observation that the ingestion of glucose administered as a glucose tolerance test in normal controls induces a marked increase in reactive oxygen species (ROS) generation by mononuclear cell (MNC) and polymorphonuclear leucocyte (PMNL) acutely with a concomitant increase in lipid peroxidation9. Since ROS generation is proinflammatory through the activation of the proinflammatory transcription factor, nuclear factor kappa B (NFkB)10, further investigations were directed at inflammatory mechanisms. Glucose intake induces an increase in key proinflammatory transcription factors, NFkB, activator protein-1 (AP-1), early growth response gene-1 (EGR1) and the inflammatory genes modulated by them: monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), matrix metalloproteinase-2 (MMP-2), MMP-9, tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1)1112. Furthermore, it was shown that saturated fat, taken as cream, also induced oxidative stress13. An infusion of triglycerides with heparin to induce an acute increase in free fatty acids (FFAs) also led to an increase in ROS generation, NFkB binding, proinflammatory cytokine, macrophage migration inhibitory factor (MIF), in addition to inducing impaired flow-mediated dilation of the brachial artery14. Such increases in FFAs are known to lead to insulin resistance as well1516. Thus, oxidative stress, inflammation and insulin resistance are closely linked. These observations led us to investigate whether a high-fat high-calorie (HFHC) meal would induce oxidative stress and inflammation. A 900 calorie fast food meal was shown to induce oxidative and inflammatory stress at the cellular and molecular level, involving the key transcription factors and genes1718. Further investigations along these lines revealed that this fatty meal induced endotoxaemia and the expression of its receptor, toll-like receptor-4 (TLR-4) and its co-receptor CD-1419. This meal also induced an increase in the expression of TNF-α and interleukin-1β (IL-1β). As stated above, TNF-α mediates insulin resistance; IL-1β, on the other hand, is a key cytokine mediating damage to the β-cell. The administration of IL-1 receptor antagonist has been shown to benefit type 2 diabetics through improved β cell function and greater insulinogenesis202122.

The acute induction of a proinflammatory milieu and several mediators of insulin resistance by the HFHC meal led to a search for non-inflammatory and anti-inflammatory meals. When the effects of the HFHC meal were compared to those of a high fruit and fibre meal, the latter was shown not to induce oxidative and inflammatory stress. Orange juice was the first food shown to be non-inflammatory in spite of all its calories being due to glucose, fructose and sucrose. This was attributed to the antioxidant and anti-inflammatory effect of flavonoids, hesperidin and naringenin, which give it its orange colour23. Further investigation demonstrated that the intake of fresh orange juice with an HFHC meal prevented oxidative and inflammatory stress, thereby increasing insulinogenesis and reducing postprandial hyperglycaemia24. Similar anti-inflammatory effects were observed with a preparation of resveratrol and grape polyphenols when administered with the HFHC meal24. Most recently, the administration of insoluble fibre with the HFHC meal has been shown to exert a similar effect on oxidative and inflammatory stress, glycaemia and insulinogenesis (unpublished observation).

The observations described above have shown that the intake of a single 900 calorie high fatty meal is a potent stimulator of oxidative stress and inflammation. In addition, it induces SOCS-3, PTP-1B and a series of serine kinases, which interfere with insulin signalling at IRS-1 level. Thus, it is clear that the cumulative effect of such meals consumed habitually is likely to result in insulin resistance and caloric overload25. In addition, since SOCS-3 interferes with leptin signalling, such meals could potentially result in lack of satiety and an increase in caloric intake2627. Progressive weight gain with insulin resistance would lead to hypertension and hypertriglyceridaemia and thus to metabolic syndrome. The data also show that a high fruit and fibre meal does not induce such changes19. Furthermore, the intake of orange juice and other fruit products results in the prevention of such changes24. Finally, the intake of fibre not only prevents oxidative stress, inflammation, endotoxaemia and the induction of TLR-4, TNF-α, IL-1β, SOCS-3 and PTP-1B but also increases insulinogenesis and reduces glycaemic excursion (unpublished observation). These observations provide the basis of strategies for a healthy lifestyle, consistent with a Mediterranean diet, known to be preventive for diabetes and atherosclerosis2829.

Insulin exerts vasodilatory and anti-inflammatory actions in addition to its profound functions as a metabolic hormone30313233. It is also relevant that insulin is a vasodilator at the macrovascular and microvascular level. This action serves to distribute glucose and other macronutrients to tissues effectively in the postprandial phase. In addition, insulin also induces the expression of endothelial nitric oxide synthase (eNOS) and the secretion of nitric oxide (NO) from the endothelium34. While it regulates the distribution, uptake and storage of macronutrients postprandially, it is also probably crucial in modulating postprandial oxidative and inflammatory stress. The concentrations at which insulin exerts the antioxidant and anti-inflammatory effects are comparable to those observed postprandially31. Thus, postprandial insulin concentrations are sufficient to prevent oxidative and inflammatory stress after an AHA meal but not an isocaloric HFHC meal since the magnitude of ROS generation, induction of inflammation and the increase in endotoxin and the expression of its receptor TLR-4 and co-receptor CD-14 are far greater19.

Insulin suppresses ROS generation, p47phox expression, intra-nuclear NFkB binding, TNF and IL-1β expression as well as MCP-1, ICAM-1, MMP-9, TF and PAI-1 concentrations when infused intravenously3132. It suppresses the expression of a series of TLRs namely, TLR-1, TLR-2, TLR-4, TLR-7, TLR-9 and the transcription factor PU.1 which regulates their transcription35. It also suppresses high mobility group box-1 (HMGB-1) expression and plasma concentrations in the obese and patients with types 2 and 1 diabetes363738. When secreted by a damaged cell, HMGB-1 functions as a proinflammatory cytokine through its binding to advanced glycation end-product (AGE) receptor. Insulin suppresses the plasma concentration of chemokines and the expression of their receptors39 and oxidative, nitrosative and inflammatory stress induced by an injection of endotoxin in normal controls36. Insulin also suppresses the proinflammatory serine kinases, IκB kinase, JNK-1 and p38MAPkinase, which cause serine phosphorylation of IRS-1 and interfere with insulin signaling37. In addition, insulin suppresses plasma FFA concentrations through the inhibition of lipolysis4041. Thus, insulin is a potential insulin sensitizer.

These comprehensive anti-inflammatory actions of insulin have led to the investigation of its potential benefits in inflammatory states. The infusion of a low dose of insulin in patients with acute myocardial infarction demonstrated that it had a suppressive effect on plasma concentrations of C-reactive protein (CRP), serum amyloid A (SAA), PAI-1, oxidative stress, creatine kinase (CK) and creatine kinase-MB (CKMB)42. Thus, in this acute clinical setting, it was anti-inflammatory, antioxidant, profibrinolytic and cardioprotective. The infusion of insulin has also been shown to suppress the expression of asthma-related genes, IL-4, CCR-2, CCR-5, transforming growth factor-β (TGF-β), LIGHT and lymphotoxin-β receptor in MNC; in addition, plasma concentrations of chemokines MCP-1 and eotaxin also fall43. Genes related to Alzheimer's disease, as expressed in MNC, have also been shown to be reduced following an insulin infusion. The genes suppressed include amyloid precursor protein, presenilins 1 and 2 and glycogen synthase kinase 3β44.

The fact that macronutrient intake is proinflammatory, in addition to inducing metabolic changes, is probably due to the fact that macronutrients are ‘external’, and when these change internal milieu significantly, inflammation ensues. However, insulin which is secreted in response to caloric intake has a major role as a metabolic regulator. The recently recognized anti-inflammatory role of insulin is linked to its metabolic role since macronutrient intake of the wrong kind and in excessive amounts can result in inflammation. This new paradigm provides the basis for the link between macronutrient intake obesity, insulin resistance and eventually, diabetes and atherosclerotic disease.