It is perhaps apparent by now that increased lipid availability can impair glucose utilization through many different mechanisms and result in the hyperglycemia of type 2 diabetes. Consequently, there are as many different approaches that can be used to design effective treatment strategies for the disease on the basis of the competition of nutrients as substrates for metabolic reactions. These strategies include limiting the availability of lipids as metabolic fuels; inhibition of fatty-acid uptake and oxidation; inhibition of gluconeogenesis; and uncoupling the energy obtained during fatty-acid oxidation with concomitant manipulation of the fatty-acid
JJ Glucose diposal and storage
Fatty acid oxidation
Gluconeogenesis ff Glucose \ ff Fatty Acids ^ ff Lipolysis
FIGURE 10.2 Summary of pathways of the glucose-fatty-acid cycle. Increased fatty-acid utilization generates intermediate substrates that impair glucose utilization while enhancing de novo glucose synthesis and storage, resulting in blood-glucose overload or hyperglycemia.
oxidation gene transcription factors peroxisome proliferator activated receptors (PPARs) (19-23).
As pointed out earlier, fatty-acid oxidation affects glycemic control not only by decreasing peripheral glucose utilization, but also by enhancing gluconeogenesis. Hence, inhibition of lipolysis is an effective strategy to reduce the availability of free fatty acids for oxidation and thus enhance glucose oxidation and decrease blood-glucose levels (19, 21). One of the early attempts to use antilipolytic agent to limit fatty-acid availability and treat type 2 diabetes was made with nicotinic acid. It was found that its inhibitory effect on lipolysis was accompanied by a stimulation of the glucose disposal. It was, however, disappointing to see that although nicotinic acid initially reduced free fatty-acid levels in type 2 diabetes, it was followed by a rebound in free fatty-acid levels that was associated with hyperglycemia and glucosuria (19). Subsequently, an analogue of nicotinic acid, acipimox, was developed, which was more potent and had less of the rebound effect than nicotinic acid (19). To date, acipimox remains an active research interest in the treatment of type 2 diabetes. In a recent experimental study with obese Zucker rats, oral administration of 150 mg/kg of acipimox significantly reduced plasma free fatty-acid (FFA), glucose, and insulin levels, and thus improved glucose tolerance while reducing insulin response (24). Clinical trials with acipimox have also yielded positive effects in the management of type 2 diabetes. In an early double-blind, placebo-controlled trial, hepatic-glucose output (HGO) and fuel use assessed by indirect calorimetry were measured in the basal state and during the last 30 minutes of a hyperglycemic clamp in obese, type 2 diabetics three times thrice during 12 hours. It was found that this protocol of prolonged suppression of lipolysis caused a reduction of fasting blood glucose and HGO while increasing peripheral hepatic sensitivity to insulin in the study subjects (25). The data from this early study were consistent with those from another overnight placebo-controlled study with acipimox (26). In the later study, 250 mg acipimox was administered three times in 12 hours to four different groups of individuals, namely lean control subjects, obese, nondiabetic individuals, obese subjects with impaired glucose tolerance, and patients with type 2 diabetes. It was found that lowering plasma FFA levels reduced insulin resis-tance/hyperinsulinemia and improved oral glucose tolerance in all groups of the study subjects (26). The observations from these studies of short-term use of acipimox have been confirmed in a randomized, double-blind, placebo-controlled study in which 25 individuals with type 2 diabetes were given 250 mg four times daily, and another 25 received a placebo for one week. The study showed that the treatment with acipimox lowered plasma FFA levels and improved acute-insulin response and insulin-mediated glucose uptake (27). However, in one of the earlier studies, acipimox gave mixed results on suppression of plasma FFA levels and had no effect on hepatic-glucose production (28). The reason for this discrepant observation with acipimox is not clear.
One strategy that has received significant attention is the use of fatty-acid oxidation inhibitors in the management of type 2 diabetes. As can be predicted from the glucose-fatty-acid cycle, the rationale in the use of this approach is that inhibiting fatty-acid oxidation would enhance peripheral tissue glucose utilization while inhibiting gluconeogenesis. Hence, there has been significant interest in the development of drugs to inhibit fatty-acid oxidation and improve glycemic control in individuals afflicted with type 2 diabetes. Long-chain fatty acids are converted to fatty acyl-CoA esters in the outer mitochondrial membrane and require carnitine to get across the inner mitochondrial membrane for oxidation in the mitochondrial matrix. The rate-limiting enzyme that catalyzes the transesterification of the fatty acyl group from Co-A to carnitine is carnitine acyltransferase 1, the predominant form being the carnitine palmitoyl transferase 1 (CPT-1). It is therefore not surprising that the first generation of fatty-acid oxidation inhibitors developed as therapeutic agents for type 2 diabetes were inhibitors of the CPT-1 enzyme (19, 21). CPT-1 inhibitors, such as etomoxir and tetradecylglycidic acid (TDGA), have the ability to decrease blood-glucose levels. However, enthusiasm for the use of this class of inhibitors of fatty-acid oxidation waned because of observations that they induced cardiac hypertrophy in rodents treated with the drugs. The next generation of CPT-1 inhibitors developed were drugs, such as SDZ and CPI 975, whose action on the liver-specific enzyme is reversible and might therefore be less toxic to the heart. Also, some monoamine oxidase inhibitors have been shown to inhibit the acylcarnitine translo-case/CPT-2 enzyme and cause reductions in blood-glucose levels (21). Further studies are required to assess both the long-term efficacy and safety of these newer compounds in human subjects.
As discussed earlier, one consequence of the catabolism of amino acids and fatty acids is the generation of metabolic substrates that are used for de novo glucose synthesis. It has been shown that hepatic insulin resistance in type 2 diabetes causes overproduction of glucose. Therefore, direct inhibition of gluconeogenesis by blocking a key enzyme in glucose synthesis, pyruvate carboxylase, or by limiting the availability of gluconeogenic substrates, is an attractive target for drug development. However, direct inhibition of pyruvate carboxylase is prone to unwanted side effects, because the enzyme has dual functions in both gluconeogenesis and in the TCA cycle. The alternative approach of using indirect inhibitors of pyruvate carboxylase that act by sequestering acetyl-CoA, thereby making it unavailable for the action of the enzyme, has shown promising results in experimental studies with animals (21). Another class of oral antidiabetic drugs, which are derivatives of acetic acid that reduce blood glucose and lipid levels without stimulating insulin secretion, has been developed. One member of this class of drugs being investigated is dichoroacetate (DCA), which inhibits hepatic glucose synthesis and stimulates glucose disposal by peripheral tissues (21, 29). A major effect of DCA is stimulation of the action of PDH, the rate-limiting enzyme in aerobic glucose oxidation, resulting in increased peripheral catabolism of alanine and lactate, thereby disrupting the Cori and alanine cycles and reducing availability of substrates for gluconeogenesis (29).
Metformin hydrochloride, a biguanide, is currently the only clinically available antidiabetic oral agent whose mechanism of action involves suppression of hepatic-glucose release through inhibition of gluconeogenesis and glycogenolysis, albeit other metabolic parameters may also be affected (21, 30, 31). For instance, it has been shown that metformin enhances insulin sensitivity at the muscle by promoting glucose transport and glycogen synthesis. It may also enhance peripheral glucose utilization by suppression of FFA release and oxidation (21). Body-weight reduction, as well as significant decreases in plasma levels of LDL cholesterol, triglycerides, and FFA, have also been reported in patients treated with metformin (21, 31). Based on these metabolic actions of metformin treatment, its use has actually been recommended as a possible strategy to prevent type 2 diabetes in individuals at high risk for developing the disease (32).
Novel pharmacologic actions of metformin have recently been described. In one study, it has been shown that the drug, in combination with a dipeptidyl peptidase IV (DPPIV) inhibitor, caused reductions in food intake and body weight while enhancing the release of GLP-1, an incretin hormone that stimulates insulin secretion and also appears to reduce appetite (33). In another study, it was shown that met-formin caused a significant decrease in mitochondrial permeability and aerobic respiration (34). However, the clinical significance of this latter in vitro observation remains to be determined.
As with any drug, the safety of metformin use has been a subject of investigation. An earlier biguanide, phenformin hydrochloride, was withdrawn from clinical use because it was associated with lactic acidosis in some patients treated with the drug. Thus far, it does not appear that lactic acidosis is a significant side effect in the treatment of uncomplicated type 2 diabetes (35, 36), albeit it could become a rare but life-threatening problem when used to treat patients with renal failure (37). A few cases of hypoglycemia have been reported in a combination therapy of met-formin and nateglinide (38).
A new concept in drug development for type 2 diabetes has emerged with the introduction of therapeutic agents that may enhance lipolysis while promoting fatty-acid oxidation in a futile cycle that does not yield metabolic energy, and thus decrease circulating FFA levels while stimulating peripheral-tissue glucose utilization (20, 24). One class of these new therapeutic agents is beta-3 agonists, which activate beta3-adrenergic receptors. An example of the beta-3 agonists currently under investigation is trecadrine, which has been reported to induce lipolysis in adipocytes, with increased oxygen consumption in white adipose tissue, while FFA levels decreased because of their utilization in nonenergy-generating tissue, such as the brown adipose tissue (39). It has also been shown that another beta-3 agonist, CL-316243, may indirectly stimulate glucose uptake in the muscle of type 2 diabetic rats by stimulating the brown adipose tissue to increase uncoupling protein content and fatty-acid oxidation, thus progressively decreasing the levels of circulating FFA (24).
The next class of agents involved in this approach of uncoupled oxidation of fatty acids is the PPARs. This is a group of three nuclear receptor isoforms encoded by different genes, PPAR-a, PPAR-S, and PPAR-y. Each of these isoforms appears to be expressed in a specific tissue because of its binding to a specific consensus DNA sequence of the peroxisome-proliferator response elements (PPREs) (22, 23, 40-42). PPAR-a is highly expressed in the liver and relatively expressed in the heart, kidney, skeletal muscle, intestinal mucosa, and brown adipose tissue (23, 42). It is the most characterized of the three PPAR subtypes and has been shown to play a prominent role in the regulation of nutrient metabolism, including fatty-acid oxidation, gluconeogenesis, and amino-acid metabolism (22, 23 , 40, 42). PPAR-S is expressed ubiquitously and has been shown to be effective in the treatment of dyslipidemia and cardiovascular disease (22). PPAR-y is mainly expressed in the brown adipose tissue where it stimulates adipogenesis and lipogenesis (22, 42).
Clinically, PPAR-a and PPAR-y agonists have been used to treat hypertriglyceridemia and insulin resistance. Thiazolidinediones are PPAR-y agonists, which enhance insulin sensitivity mainly at the skeletal muscle and adipose tissue, with some effect at the liver where they increase insulin-stimulated glucose disposal (21). These drugs increase triglyceride uptake into the adipose tissue, thereby reducing circulating FFA levels. One member of the PPAR-y agonists group of drugs that had been previously used clinically is troglitazone. In one study, a randomized placebo-controlled trial was performed with troglitazone to determine its effect on whole-body insulin sensitivity, pancreatic P-cell function, and glucose tolerance in Latino women with impaired glucose tolerance and a history of gestational diabetes (43). After baseline oral glucose tolerance (OGTT) and intravenous glucose tolerance (IVGTT) tests, each of three groups (14/group) of subjects was assigned to receive one of three treatments, a placebo, 200 mg troglitazone, or 400 mg troglitazone daily for 12 weeks. It was found that insulin sensitivity assessed by the minimal model analysis of IVGTT results changed by only 4 percent in the placebo group but was increased by 40 percent and 88 percent above basal in the groups treated with 200 mg and 400 mg troglitazone, respectively. Troglitazone treatment was also associated with a dose-dependent reduction in the total insulin output during the glucose-tolerance tests (43). Another PPAR-y agonist that is still clinically available is pioglitazone, whose potency has been compared with that of troglitazone with reference to its effects as inhibitors of fatty oxidation, esterfication, and gluconeo-genesis (44). It was concluded that at similar concentration, troglitazone was more effective than pioglitazone in inhibiting fatty-acid oxidation and gluconeogenesis, and that the inhibition of gluconeogenesis by troglitazone may be the result of its inhibition of fatty-acid oxidation (44). Unfortunately, troglitazone has been withdrawn from clinical use because of its hepatotoxicity, leaving pioglitazone and rosiglitazone as the only two thiazolidinediones currently available for clinical use (21).
The PPAR-a agonist group of drugs include those synthetic, therapeutic agents that are molecular targets for fibrates, such as gemfibrozil, bezafibrate, clofibrate, and fenfofibrate, which are used to treat dyslipidemia and cardiovascular disease. PPAR-a promotes fatty-acid transport across cell membranes and converts them into a metabolic form that precedes their subsequent metabolism. These drugs are gaining popularity in combination treatment with the statins (23).
A nutritional therapy for type 2 diabetes based on nutrient competition as metabolic substrates is an approach that has been examined in studies performed by our group and others. In a series of studies, it had been shown that fatty acids stimulate insulin secretion through a mechanism involving fatty-acid oxidation (45-49), while the amino acid L-glutamine inhibits insulin secretion (50). It had also been previously shown that L-glutamine inhibits fatty-acid oxidation by islets, because it is a preferred fuel source for these cells (51). We subsequently showed that addition of L-glutamine to a fatty acid perifusate inhibited fatty-acid oxidation and prevented fatty acid-induced desensitization of islet response to glucose stimulation (52). Based on these in vitro observations, we hypothesized that L-glutamine supplementation during high-fat feeding would prevent insulin resistance characterized by hyperinsuline-mia and hyperglycemia, as seen in C57BL/6J (B/6J) mice. In the B/6J mice, high-fat feeding causes obesity associated with type 2 diabetes (53, 54).
In our L-glutamine supplementation studies, the effect of L-alanine on glucose dysregulation induced by high-fat feeding was also examined. Each of four groups of 10 age- and weight-matched male B/6J was raised on one of four diets: 1) low fat, low sucrose (LL); 2) high fat, low sucrose alone (HL); 3) high fat, low sucrose supplemented with L-glutamine; and 4) high fat, low sucrose supplemented with L-alanine. Food intake, body weight, and plasma-glucose and insulin levels were monitored over time. We found no difference in food intake per unit body weight between the groups after the first two weeks of feeding. However, the mean body weight of the LL group measured at 16 weeks was significantly lower than that of the HL group, as shown in Figure 10.3. Although supplementation with each of the
10 12 14 16
10 12 14 16
FIGURE 10.3 Body weights of B/6J mice fed a low-fat diet or a high-fat diet with or without supplemental Gln or Ala. Values are means ± SEM, n = 10. The error bars were so small that they are invisible in most data points. Means at a particular time point with different letters are significantly different, p < 0.05. Abbreviations used: HL, high fat, low sucrose; HL + Gln, high fat, low sucrose with L-glutamine supplementation; HL + Ala, high fat, low sucrose with L-alanine supplementation; LL, low fat, low sucrose. Reprinted with permission from Figure 2 of reference 55.
amino acids caused 10 percent reduction in body weight compared with HL feeding, only L-glutamine supplementation resulted in persistent reductions in plasma-glucose and insulin levels during the 5.5-month duration of study. We also found that when L-glutamine was added to the HL diet of obese hyperglycemic and hyperin-sulinemic animals for two months, body-weight gain, shown in Figure 10.4, as well as hyperglycemia and hyperinsulinemia, were all significantly attenuated (55). In another study, we also found that L-glutamine supplementation prevents impaired glucose regulation associated with hyperlipidemia induced by intravenous lipid administration (56). These observations have been confirmed in a study by other investigators who found that parenteral glutamine supplementation augmented whole-body insulin stimulation of whole-body glucose utilization, thus suggesting improved insulin sensitivity (57). It is of interest that glycogen synthesis is stimulated by L-glutamine (58-61), which, as previously noted, inhibits fatty-acid oxidation (51) and lipolysis (62, 63) while stimulating lipogenesis (64). The enhancement of glycogen synthesis by glutamine would imply an increase in gluconeogenic fluxes that is associated with increased rate of nonoxidative glucose clearance (65) that may result in near-normal blood-glucose levels during high-fat feeding, as seen in our studies. The clinical applicability of this nutritionally based therapeutic approach remains to be evaluated, albeit the clinical implication of our observations using this approach is discussed in the chapter in this book by Drs. Lien and Feinglos.
0 2 6 9 13 16 20 23 27 30 37 44 58 Days After Diet Change
0 2 6 9 13 16 20 23 27 30 37 44 58 Days After Diet Change
FIGURE 10.4 Effect of supplemental glutamine on relative body weight in heavy, hyperglycemic adult mice switched from the high-fat (HL) to the HL + Gln diet. The gap in the x-axis denotes an alteration in the scale because of the different intervals of determination. Two groups of mice (n = 10/group) were fed the HL diet for four months before one group was switched to the HL + Gln diet for two months, during which time the other continued being fed the HL diet. Different letters indicate significant differences between groups. Values are mean ± SEM, n = 10. Reprinted with permission from Figure 5 of reference 55.
It is quite clear that the processing of nutrients is intertwined through intermediary metabolism in the body. Consequently, it is imperative that there is a natural competition among the key nutrients, carbohydrate, fatty acids, and amino acids as sources of biological energy. Which nutrient becomes a primary source of energy is regulated by the nutritional status (fed or fasted state) with the concomitant hormonal balance. The convergence of the metabolism of nutrients in the tricarbox-ylic-acid cycle has very obvious implications in the utilization of each nutrient as substrates for the generation of expendable energy or the synthesis of macromole-cules as storage forms of energy or for tissue maintenance.
Depending upon the nutritional status, various studies have shown that the predominant energy-yielding sources are glucose and fatty acids, which compete with each other as sources of metabolic energy. There is overwhelming evidence to show that the converging metabolism of these two nutrients in the glucose-fatty-acid cycle plays a predominant role in the pathogenesis of type 2 diabetes, albeit some studies have challenged the view that this cycle contributes to insulin resistance (66, 67). However, based on the hypothesis of the glucose-fatty-acid cycle, it is possible to design effective therapeutic strategies for type 2 diabetes mellitus. These strategies for drug development could involve inhibitions of fatty-acid oxidation and gluconeogenesis, manipulation of the enzymatic processes involved in nutrient processing, molecular targets for metabolic engineering, or a simple nutritional approach. A significant number of effective therapeutic agents have been developed, but there is still enormous potential for drug development for diabetes treatment on the basis of metabolic interactions of nutrients. This review that has summarized these nutrient interactions thus provides a valid scientific basis for effective strategies in designing new therapeutic agents for type 2 diabetes.
II Type 2 Diabetes in Childhood: Diagnosis, Pathogenesis, Prevention, and Treatment
Michael Freemark, M.D.
III Risk Factors and the Pathogenesis of Insulin Resistance and
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