The contribution of PPARγ activity in specific tissues to whole-body insulin sensitivity has been difficult to dissect. Although PPARγ levels are highest in white adipose tissue, multiple insulin-sensitive tissues express PPARγ and thus in theory might contribute to the effects of TZDs on whole-body insulin sensitivity. Furthermore, the endogenous role of PPARγ in tissues has been difficult to determine since global genetic KO of PPARγ in the mouse results in embryonic lethality by embryonic day 10 (38
), and mice with heterozygous deficiency of PPARγ display improved insulin sensitivity (39
). In humans, specific PPARγ mutations have been shown to lead to obesity (41
) or marked insulin resistance (42
), and a common polymorphism (Pro12Ala) is associated with improved insulin sensitivity (43
). To explore the role of muscle PPARγ in insulin sensitivity and glucose homeostasis, we generated muscle-specific PPARγ–deficient mice using a Cre/loxP
We find that mice with muscle-specific disruption of PPARγ have normal glucose homeostasis and insulin levels, including normal responses to glucose or pharmacologic insulin challenge. The MuPPARγKO mice, however, have significant whole-body insulin resistance when measured directly using the hyperinsulinemic-euglycemic clamp. This mild insulin-resistance phenotype is similar to that of the muscle-specific insulin receptor KO (MIRKO) mouse, which has normal glucose and insulin levels and normal responses to glucose or insulin challenge (20
), yet has insulin resistance when measured by euglycemic-hyperinsulinemic clamp (26
). In MIRKO mice, this is associated with severe impairment of insulin-stimulated glucose uptake in muscle and compensatory increased glucose uptake in fat due to undefined tissue crosstalk (26
). In contrast, in MuPPARγKO mice the whole-body insulin resistance is localized to nonmuscle tissues, including the liver and, possibly, fat.
Related results have been observed in mice with selective KO of glucose transporter-4 in muscle (M-G4KO). These mice have severe loss of basal, insulin-stimulated, and contraction-stimulated glucose uptake in skeletal muscle (32
) and have whole-body insulin resistance as measured by euglycemic-hyperinsulinemic clamp (45
). The glucose homeostasis defect in M-G4KO mice is more severe, and they develop secondary insulin resistance in liver and adipose tissue mediated by glucose toxicity (45
). Since MuPPARγKO mice have normal glucose levels and normal insulin-stimulated glucose uptake in muscle, it is likely that a mechanism other than glucose toxicity is responsible for the insulin resistance that develops in the liver and, possibly, fat of these mice.
The hepatic insulin resistance in MuPPARγKO mice might be secondary to altered adipokine release associated with increased adiposity. Consistent with this possibility, preliminary studies suggest altered adipokine expression in adipose tissue as determined by quantitative RT-PCR. The expression of ACRP30 was reduced 55% in adipose tissue from MuPPARγKO mice compared with controls (P
= 0.09), while the expression of TNF-α was increased 63% (P
= NS) in adipose tissue from MuPPARγKO mice (unpublished data). Both of these trends could be expected to induce hepatic insulin resistance (46
), though proof of causality in our model will require additional experimentation.
The disruption of muscle PPARγ in mice leads to excess adiposity. This phenotype is similar to that of MIRKO mice, which also have normal body weight on normal chow but develop increased fat depot mass (20
). In MIRKO mice, however, this is accompanied by increased insulin-stimulated glucose uptake in adipose tissue. Since MuPPARγKO mice have a tendency for diminished responsiveness to insulin in adipose tissue, it is unlikely that MIRKO and MuPPARγKO mice develop enlarged fat pads via the same mechanisms.
The excess weight gain and adiposity observed in MuPPARγKO mice on a high-fat diet occurred despite a reduction in caloric intake. This suggests that the adiposity in MuPPARγKO mice is not due to a defect in appetite regulation such as would occur in leptin resistance. In fact, the decrement in food intake associated with increased adiposity, coupled with the observed trend toward higher serum leptin levels, is consistent with an intact leptin response in MuPPARγKO mice. The feeding data instead suggest that the weight gain/adiposity of MuPPARγKO mice may be due to higher energy efficiency, that is, reduced fuel oxidation and increased fuel storage. Given that skeletal muscle is a major site of fuel oxidation (48
), it is possible that loss of muscle PPARγ could produce such an effect. A preliminary indirect calorimetry study has revealed no differences in oxygen consumption, CO2
production, and physical activity levels between MuPPARγKO and Lox control mice on a high-fat diet (unpublished data). Likewise, we found no significant differences in uncoupling protein-1, -2, or -3 expression between MuPPARγKO and Lox muscle, as represented on the U74Av2 microarray (unpublished data).
An alternative explanation for excess fat mass in MuPPARγKO mice would be an impaired ability of skeletal muscle to use lipid fuel substrate leading to a shunting of lipid to adipose tissue. Indeed, it is postulated that PPARγ mediates upregulation of lipid use in skeletal muscle (49
), in a manner similar to the essential role of isoform PPARα in the upregulation of lipid oxidation in cardiac muscle (52
). Though isoform PPARα is expressed in skeletal muscle at levels higher than PPARγ (ref. 54
; our unpublished data), there is little to no impairment in skeletal muscle lipid utilization gene regulation upon loss of PPARα (55
). This suggests that other factors, perhaps PPARγ, play an important role. Consistent with this possibility, there are differences in lipid metabolism gene expression in MuPPARγKO muscle.
Defects in use of fatty acids by skeletal muscle have also been postulated to contribute to the development of type 2 diabetes (56
), such that excess delivery of lipid to liver and adipose tissues leads to insulin resistance in these tissues (58
). This represents an alternative mechanism that could account for the diminished insulin sensitivity in liver and adipose tissue in MuPPARγKO mice, though we did not detect differences in lipid content of serum or tissues of MuPPARγKO mice other than increased adipose mass.
TZDs improve whole-body glucose homeostasis by increasing insulin-stimulated glucose uptake in skeletal muscle (5
). This action on muscle may be direct (16
) or indirect through the effects of TZDs on fat (13
). It is thus informative that although MuPPARγKO mice develop insulin resistance at baseline, they nonetheless have a normal response to RSG with a reduction in hyperinsulinemia and improved whole-body glucose homeostasis. Taken together, these improvements are very suggestive of increased insulin sensitivity, though in the absence of clamp data from TZD-treated MuPPARγKO mice it remains possible that subtle deficiencies in the TZD response of MuPPARγKO mice exist. The normal response in multiple parameters of MuPPARγKO mice to TZDs, however, supports the hypothesis that PPARγ agonists improve insulin sensitivity by direct actions on tissues other than skeletal muscle, presumably adipose tissue. This hypothesis has been suggested in a previous study where TZDs failed to improve glucose homeostasis in mice with severe lipoatrophy (15
). Adipose tissue secretes a number of factors, including leptin, TNF-α, FFAs, resistin, IL-6, and ACRP30, which modulate insulin sensitivity (59
). TZDs can induce beneficial changes in the adipose production of most of these, including lower FFAs (60
), TNF-α (61
), and resistin (62
) levels, as well as increased ACRP30 levels (63
). These salutary changes are accompanied by a reduction of adipocyte size, which is also expected to improve overall body-glucose homeostasis (64
). Any or a combination of these factors could represent a mechanism for the insulin-sensitizing effects of TZDs on skeletal muscle by adipose tissue.
Our data, as well as other recent studies, suggest that PPARγ in skeletal muscle may directly modulate expression of some genes of lipid metabolism. In normal human skeletal muscle, Lapsys et al. (37
) found a strong positive correlation between the expression level of PPARγ and the mRNA levels of LPL, FABP3, and mCPT1b. In contrast, we observed an increase of FABP3 in MuPPARγKO mice, but no change in LPL or mCPT1b. We did observe, however, changes in expression of LC-FACS2 and FATP1 in skeletal muscle of MuPPARγKO mice. Both of these genes are regulated in response to PPAR activity in liver (65
), but in a positive manner — the opposite of the effect we observed. These findings reinforce the suggestion by Way et al. (51
) that PPARγ activity in skeletal muscle may have different effects on gene expression compared with adipose tissue or liver. An in-depth analysis of gene expression in muscle of these mice before and after TZD treatment should allow definition of the genes regulated by PPARγ in skeletal muscle.
In summary, disruption of muscle PPARγ leads to increased fat mass, as well as hepatic insulin resistance. Disruption of muscle PPARγ, however, does not block the beneficial effects of TZDs on glucose homeostasis. These data indicate that the insulin-sensitizing effects of TZDs on muscle are indirect, but that PPARγ in muscle plays a role in regulation of whole-body lipid storage and hepatic insulin sensitivity through tissue crosstalk, perhaps mediated by alterations in regulation of muscle lipid metabolism genes.