Insulin resistance and diabetes have profound effects on hepatic carbohydrate and lipid metabolism. In vivo hepatic fat oxidation was severely impaired in the fasted 12-week-old ZDF rat, consistent with the previous reports of increased de novo lipogenesis in the fed state (35
). By 22 weeks, hepatic fat oxidation index in the ZDF rat was no longer impaired but remained dysfunctional with regard to the distribution between ketogenesis (twofold lower than normal) and TCA cycle oxidation (twofold higher than normal), suggesting a reorganization of mitochondrial fat oxidation with the onset of insulinopenia. Hepatic glucose production in diabetic rats was also remarkably age dependent. Together with previous work, the current data indicate that elevated fasting glucose production in the ZDF rat occurs initially as a consequence of increased glycogen breakdown (28
), followed shortly by increased conversion of glycerol to glucose (22
), and then, in later stages of the phenotype, it occurs due to increased gluconeogenesis from substrates like lactate and amino acids (). However, other studies have revealed less remarkable changes in the sources of glucose production (37
), perhaps due to differences in fasting times or methodological approaches.
Abnormal mitochondrial metabolism is a key feature of insulin-resistant skeletal muscle (17
) and has been implicated in human insulin-resistant liver (15
). However, there are few in vivo data regarding mitochondrial metabolism in insulin-resistant liver. Here, FAO gene expressions, including PGC-1α were not robustly altered, indicating that defects in hepatic mitochondrial fat oxidation may be metabolically mediated. Impaired fat oxidation in hepatocytes of nondiabetic Zucker fatty rats (38
) and ZDF rats (39
) has been attributed to increased levels of malonyl-CoA (39
) and inhibition of CPT-1–mediated transport of long-chain fatty acids into mitochondria (40
). Liver mitochondria from nondiabetic Zucker fatty rats may (40
) or may not (41
) have a primary defect in oxidative capacity. In humans, abnormal mitochondrial respiratory chain activity is associated with nonalcoholic fatty liver disease (16
). In any case, defects in hepatic energy generation seem inconsistent with the increased energy requirements of excessive hepatic gluconeogenesis and lipogenesis associated with hepatic insulin resistance and diabetes.
) and animal models (13
) with primary defects in hepatic fat oxidation become hypoglycemic, yet the ZDF rat has elevated fasting glucose production despite impaired fat oxidation. This is possible because elevated glucose production in the ZDF liver comes largely from the nonenergy demanding pathways of glycogen breakdown and conversion of glycerol to glucose (GNGglycerol
) (). The former process is essentially energy neutral, while the latter process contributes to net energy production by way of NADH generated in the α-glycerophosphate dehydrogenase step. Additionally, although mitochondrial metabolism is dysfunctional in the diabetic liver, only total β-oxidation and ketogenesis are impaired; the mitochondrial pathways of pyruvate carboxylase, α-glycerophosphate dehydrogenase, and the TCA cycle are, in fact, elevated. The inappropriate segregation of β-oxidation products toward oxidation is reminiscent but seemingly opposite to mitochondrial metabolism in insulin-resistant skeletal muscle, where fatty acid overload induces fat oxidation but results in the build-up of acyl-carnitine/CoA intermediates (20
) due to impaired TCA cycle flux (18
A reasonable response to impaired hepatic fat oxidation is to correct the condition by pharmacological intervention. While PPAR-α agonists (i.e., WY14,643 and fibrate drugs) stimulate fat oxidation and improve insulin resistance, they do not always improve glycemia and/or endogenous glucose production in diabetic rodent models (29
) or humans (42
). Here, WY14,643 stimulated hepatic β-oxidation in diabetic rats by overinduction of TCA cycle flux, even at a relatively low dose (one-third the typical rodent dose), and also ketogenesis at a higher dose (typical rodent dose). Concurrently, hepatic pyruvate carboxylase flux was stimulated by WY14,643 treatment, and although much of the effect was dissipated by an induction of pyruvate cycling, GNG tended to be increased rather than decreased (). Moreover, hepatic gluconeogenesis was increased in lean control animals treated with WY14,643, reinforcing the indication that induction of hepatic fat oxidation stimulates hepatic glucose production. These data do not diminish the utility of PPAR-α agonist drugs, which are commonly used to treat hyperlipidemia, but rather highlight an unanticipated effect on liver metabolism that may go unnoticed because improved insulin sensitivity can metabolically supersede the adverse effect of stimulated gluconeogenesis on glycemia. This may be particularly true in humans, where hepatic PPAR-α expression is less abundant than in rodents (43
It is unclear whether paradoxically increased FGF-21 expression in the hypoketotic liver of ZDF rats and other diabetic rodents (44
) is due to a PPAR-α–related defect or some other form of resistance to the paracrine effects of FGF-21. However, increased lipolysis and circulating NEFAs in these animals suggests that FGF-21's endocrine effects on adipose tissue (32
) remain intact. Further studies are required to determine whether overproduction of FGF-21 by the liver is a diabetogenic feature meant to compensate for impaired fat oxidation and whether this also contributes to hyperlipidemia by exacerbating the lipolytic state of insulin-resistant adipose.
Methodological considerations and limitations.
Measurements of ketogenesis by ketone tracer dilution may be vulnerable to overestimation via extrahepatic exchange processes (45
), termed pseudoketogenesis (46
). This was demonstrated in hepatectomized dogs given a bolus of ketone tracers and the pyruvate dehydrogenase activator trichloroacetate (47
). However, others showed that steady-state infusion of low enrichments of ketone tracers matched the “gold standard” of hepatic ketone A/V difference in both fasted normal and diabetic dogs (25
). We cannot rule out the possibility that the method overestimated ketogenesis in the rat, but we consider it unlikely that the approach would underestimate ketone turnover in diabetic rats compared with controls. Most importantly, the data correctly predict changes in hepatic fat metabolism after interventions (i.e., fasting, feeding, etomoxir treatment, and octanoate infusion; see supplemental data, online appendix).
With regard to impaired hepatic fat oxidation in the ZDF rat, it is unclear whether this finding is a general feature of obesity and insulin resistance or a defect specific to the absence of a functioning leptin signaling pathway (49
). Thus, the hepatic fluxes should also be studied in non–leptin-based rodent models to understand more clearly the role of these defects in the insulin-resistant liver. Moreover, the approaches used here are completely translatable to human subjects and will be valuable tools for probing fluxes in the liver during metabolic pathophysiologies and/or drug therapies.
These data reveal abnormal mitochondrial metabolism in the ZDF rat liver leading to inefficient fat oxidation, a process known to interfere with insulin signaling in muscle (50
); but induction of other mitochondrial pathways (TCA cycle flux and pyruvate carboxylase) reveals a complex defect in mitochondrial metabolism in the liver during diabetes. PPAR-α agonist treatment lowered insulin and NEFA levels and improved mitochondrial ketogenesis and total fat oxidation in diabetic rats but also induced the mitochondrial fluxes of pyruvate carboxylase and TCA cycle flux and the stimulation of gluconeogenesis. Future studies on other models of insulin resistance and in human subjects will help to determine whether defects in hepatic mitochondrial metabolism are a universal feature of insulin resistance.