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ROLE OF THE ADIPOCYTE IN THE PATHOGENESIS OF TYPE 2 DIABETES MELLITUS: THE HARMONIOUS QUARTET

The majority (>80%) of type 2 diabetics in the US are overweight (221). Both lean and especially obese type 2 diabetics are characterized by day-long elevations in the plasma free fatty acid concentration, which fail to suppress normally following ingestion of a mixed meal or oral glucose load (222). Free fatty acids (FFA) are stored as triglycerides in adipocytes and serve as an important energy source during conditions of fasting. Insulin is a potent inhibitor of lipolysis, and restrains the release of FFA from the adipocyte by inhibiting the enzyme hormone sensitive lipase. In type 2 diabetics the ability of insulin to inhibit lipolysis (as reflected by impaired suppression of radioactive palmitate turnover) and reduce the plasma FFA concentration is markedly reduced (17). It is now recognized that chronically elevated plasma FFA concentrations can lead to insulin resistance in muscle and liver (1,4,19,21,22,51,162,223,224) and impair insulin secretion (22,225,226) . Thus, elevated plasma FFA levels can cause/aggravate the three major pathogenic disturbances that are responsible for impaired glucose homeostasis in type 2 diabetic individuals and the time has arrived for the "triumvirate" (muscle, liver, beta cell) to be joined by the "fourth musketeer" (227) to form the "harmonious quartet" . In addition to FFA that circulate in plasma in increased amounts, type 2 diabetic and obese nondiabetic individuals have increased stores of triglycerides in muscle (228,229) and liver (230,231) and the increased fat content correlates closely with the presence of insulin resistance in these tissues. Triglycerides in liver and muscle are in a state of constant turnover and the metabolites (i.e., fatty acyl CoAs) of intracellular FFAs have been shown to impair insulin action in both liver and muscle (1,4,92) . This sequences of events has been referred to as "lipotoxicity" (1,4,22,93). Evidence also has accumulated to implicate "lipotoxicity" as an important cause of beta cell dysfunction (22,93) (see earlier discussion).

FFA and Muscle Glucose Metabolism

Four decades ago, Randle (232) proposed that increased FFA oxidation restrains glucose oxidation in muscle by altering the redox potential of the cell and by inhibiting key glycolytic enzymes . The excessive FFA oxidation: (i) leads to the intracellular accumulation of acetyl CoA, a potent inhibitor of pyruvate dehydrogenase (PDH), (ii) increases the NADH/NAD ratio, causing a slowing of the Krebs cycle, and (iii) results in the accumulation of citrate, a powerful inhibitor of phosphofructokinase (PFK). Inhibition of PFK leads to the accumulation of glucose-6-phosphate (G-6-P) which in turn inhibits hexokinase II. The block in glucose phosphorylation causes a build up of intracellular free glucose which restrains glucose transport into the cell via the GLUT4 transporter. The resultant decrease in glucose transport was postulated to account for the impairment in glycogen synthesis, although a direct inhibitory effect of fatty acyl CoAs on glycogen synthase also has been demonstrated (233). This sequence of events via which accelerated plasma FFA oxidation inhibits muscle glucose transport, glucose oxidation, and glycogen synthesis is referred to as the "Randle Cycle" (232). It should be noted that the same scenario would ensue if the FFA were derived from triglycerides stored in muscle (228,229) or from plasma (222).
Felber and coworkers (59,159,162,234,235) were amongst the first to demonstrate that in obese non-diabetic and diabetic humans, basal plasma FFA levels and lipid oxidation (measured by indirect calorimetry) are increased and fail to suppress normally after glucose ingestion. The elevated basal rate of lipid oxidation was strongly correlated with a decreased basal rate of glucose oxidation, as well as with reduced rates of glucose oxidation and non-oxidative glucose disposal (glycogen synthesis) following ingestion of a glucose load. Further validation of the Randle Cycle in man has come from studies employing the euglycemic insulin clamp. In normal subjects, physiologic hyperinsulinemia (80-100 uU/ml) causes a 60-70% decline in plasma FFA concentration and a parallel decline in plasma FFA and total body lipid oxidation (18). When Intralipid is infused concomitantly with insulin to maintain or increase the plasma FFA concentration/oxidation, both glucose oxidation and non-oxidative glucose disposal are inhibited in a dose dependent fashion (223). Using magnetic resonance imaging, it has been shown that the FFA-induced inhibition of non-oxidative glucose disposal reflects impaired glycogen synthesis (236). The inhibitory effect of elevated plasma FFA levels can be observed at all plasma insulin concentrations, spawning the physiologic and pharmacologic range (223).
The inhibitory effect of an acute elevation in plasma FFA concentration on muscle glucose metabolism is time dependent. Thus, the earliest (within 2 hours) observed abnormality is a defect in glucose oxidation (237), as would be predicted by operation of the Randle cycle (232). This is followed (between 2-3 hours) by defects in glucose transport and phosphorylation and eventually (after 3-4 hours) by impaired glycogen synthesis.

Randle Cycle Revisited: Biochemical/Molecular Basis Of FFA-Induced Insulin Resistance

The original description of the Randle cycle was formulated based upon experiments performed in rat diaphragm and heart muscle (232). More recent studies performed in human skeletal muscle suggest that mechanisms in addition to those originally proposed by Randle are involved in the FFA-induced insulin resistance. Thus, several groups (236,238,239) have failed to observe a rise in muscle G-6-P and citrate concentrations when insulin-stimulated glucose metabolism was inhibited by an increase in the plasma FFA concentration. Elevated plasma FFA levels also failed to inhibit muscle phosphofructokinase activity. Thus, while increased FFA/lipid oxidation and decreased glucose oxidation are closely coupled, as originally demonstrated by Randle, mechanisms other than product (i.e., elevated intracellular G-6-P and free glucose concentrations) inhibition of the early steps of glucose metabolism must be invoked to explain the defects in glucose transport, glucose phosphorylation and glycogen synthesis.
Studies in humans and animals have shown a strong inverse correlation between insulin-stimulated glucose metabolism and increased intramuscular lipid pools, including triglyceride (240-242), diacylglycerol (DAG) (243,244), and long chain fatty acyl CoAs (FA-CoA) (245). An acute elevation in plasma FFA concentration leads to an increase in muscle fatty acyl CoA and DAG concentrations. Both long chain fatty acyl CoAs and DAG activate PKC theta (243), which increases serine phosphorylation with subsequent inhibition of IRS-1 tyrosine phosphorylation (246,247). Consistent with this observation, two groups have shown that in human muscle elevated plasma FFA levels inhibit insulin-stimulated tyrosine phosphorylation of IRS-1, the association of the p85 subunit of PI-3 kinase with IRS-1, and activation of PI-3-kinase (248,249) . Direct effects of long chain fatty acyl CoAs on glucose transport (250), glucose phosphorylation (251), and glycogen synthase (233) also have been demonstrated in muscle . Lastly, increased muscle ceramide levels (secondary to increased long chain fatty acyl CoAs) have been shown to interfere with glucose transport and to inhibit glycogen synthase in muscle via activation of PKB (252). In summary, elevated plasma FFA concentrations can induce insulin resistance in muscle via multiple mechanisms involving alterations in a variety of intracellular lipid signaling molecules which exert their inhibitory effects on multiple steps (insulin signal transduction system, glucose transport, glucose phosphorylation, glycogen synthase, pyruvate dehydrogenase, Krebs cycle) involved in glucose metabolism.

FFA and Blood Flow

Insulin is a vasodilatory hormone and the stimulatory effect of insulin on muscle glucose metabolism has been shown to result from: (i) a direct action of insulin to augment muscle glucose metabolism, and (ii) increased blood flow to muscle (253,254). The vasodilatory effect of insulin is mediated via the release of nitric oxide from the vascular endothelium (255). In insulin resistant conditions, such as obesity and type 2 diabetes, some investigators have suggested that as much as half of the impairment in insulin-mediated whole body and leg muscle glucose uptake is related to a defect in insulin's vasodilatory action (253,254) , although the link between insulin-mediated vasodilation and increased blood flow has been challenged by others (256).
Because type 2 diabetes and obesity are insulin resistant states characterized by day-long elevation in the plasma FFA concentration (222) and impaired endothelium dependent vasodilation (253), investigators have examined the effect of increased plasma FFA levels on limb blood flow and muscle glucose uptake (257,258). In healthy, non-diabetic subjects an acute physiologic increase in plasma FFA concentration inhibited methacholine (endothelium dependent) but not nitroprusside (endothelium independent) stimulated blood flow in association with an impairment in insulin-stimulated muscle glucose disposal. In subsequent studies, the inhibitory effect of FFA on insulin-stimulated leg blood flow was shown to be associated with decreased nitric oxide production (259). FFA elevation also inhibits nitric oxide production in endothelial cell cultures by decreasing nitric oxide synthase activity (259). Since the IRS-1/PI-3 kinase signal transduction pathway is involved in the regulation of nitric oxide synthase activity (260), one could hypothesize that FFA-induced inhibition of the insulin signal transduction pathway is responsible for the blunted vasodilatory response to the hormone.

FFA and Hepatic Glucose Metabolism

The liver plays a pivotal role in the regulation of glucose metabolism (1,4,6,11,16,205). Following carbohydrate ingestion, the liver suppresses its basal rate of glucose production takes up approximately one-third of the glucose in the ingested meal (12,24,25,205). Collectively, suppression of hepatic glucose production and augmentation of hepatic glucose uptake account for the maintenance of about half of the rise in plasma glucose concentration following ingestion of a carbohydrate meal.
Hepatic glucose production is regulated by a number of factors , of which insulin (inhibits HGP) and glucagon and FFA (stimulate HGP) are the most important. In vitro studies have demonstrated that plasma FFA are potent stimulators of HGP and do so by increasing the activity of pyruvate carboxylase and phosphoenolpyruvate carboxykinase, the rate limiting enzymes for gluconeogenesis (261,262). FFA also enhance the activity of glucose-6-phosphatase, the enzyme that ultimately controls the release of glucose by the liver (263).
In normal subjects, increase plasma FFA levels stimulate gluconeogenesis (264,265), while a decrease in plasma FFA concentration reduces gluconeogenesis (264). It has documented that a significant portion of the suppressive effect of insulin on hepatic glucose production is mediated via inhibition of lipolysis and a reduction in circulating plasma FFA concentrations (16,266,267). Moreover, FFA infusion in normal humans under conditions that simulate the diabetic state (268) and in obese insulin-resistant subjects (269) enhances hepatic glucose production, most likely secondarily to stimulation of gluconeogenesis.
In type 2 diabetic subjects, the fasting plasma FFA concentration and lipid oxidation rate are increased and are strongly correlated with both the elevated fasting plasma glucose concentration and basal rate of hepatic glucose production (18,51,59,162,190,270). The relationship between elevated plasma FFA concentration, FFA oxidation, and hepatic glucose production in obesity and type 2 diabetes is explained as follows: (i) increased plasma FFA levels, by mass action, augment FFA uptake by hepatocytes, leading to accelerated lipid oxidation and accumulation of acetyl CoA. The increased concentration of acetyl CoA stimulates pyruvate carboxylase, the rate limiting enzyme in gluconeogenesis (261,262), as well as glucose-6-phosphatase, the rate-controlling enzyme for glucose release from the hepatocyte (263); (ii) the increased rate of FFA oxidation provides a continuing source of energy (in the form of ATP) and reduced nucleotides (NADH) to drive gluconeogenesis; (iii) elevated plasma FFA induce hepatic insulin resistance by inhibiting the insulin signal transduction system (244-248). In type 2 diabetic patients these deleterious effects of elevated plasma FFA concentrations occur in concert with increased plasma glucagon levels (181,190,271), increased hepatic sensitivity to glucagon, and increased hepatic uptake of circulating gluconeogenic precursors.

Summary: FFA and the Pathogenesis of Obesity and Type 2 Diabetes Mellitus

In obese individuals and in the majority (>80%) of type 2 diabetic subjects, there is an expanded fat cell mass and the adipocytes are resistant to the antilipolytic effects of insulin (18). Most obese and diabetic individuals are characterized by visceral adipocity (272) and visceral fat cells have a high lipolytic rate, which is especially refractory to insulin (273). Not surprisingly, both type 2 diabetes and obesity are characterized by an elevation in the mean day-long plasma FFA concentration. Elevated plasma FFA levels, as well as increased triglyceride/fatty acyl CoA content in muscle, liver, and beta cell, lead to the development of muscle/hepatic insulin resistance and impaired insulin secretion.