Insulin and IGF-1 act through highly homologous receptors to initiate their functions on metabolism and growth. While insulin and IGF-1 can have distinct physiological roles, at the cellular level they regulate many of the same signaling pathways. We have previously shown that the IR and IGFR have similar effects in regulating gene expression in preadipocytes, with the main difference between insulin and IGF-1 effects being due to a modulation in amplitude of the signal created by the specific ligand-receptor interaction 12
. To understand the role of complementary actions of insulin and IGF-1 in fat metabolism and development, we created mice with a combined knockout of IR and IGFR in fat using Cre recombinase under the control of the adipocyte specific aP2 promoter. The resulting FIGIRKO mice exhibit reduced body weight, reduced white adipocyte number and an almost complete absence of brown adipose tissue with dramatic sensitivity to cold exposure. Nonetheless, these mice are completely protected from age- and HFD-induced obesity and glucose intolerance, at least in part, due to increased energy expenditure and increased glucose uptake in skeletal muscle.
From the data, it appears that these changes occurred with only a partial knockdown of IR and IGF1R, since the mRNA levels of these two receptors in different fat depots were reduced only 30 to 60% in FIGIRKO mice. However, this is almost certainly an underestimate of the efficiency of the knockout, since inactivation of both IR and IGF1R dramatically impairs adipocyte differentiation, and in vivo, we can only assess the degree of inactivation in the residual adipose tissue in which the cells may have recombined some, but not all four, targeted alleles. This marked impairment of adipose tissue development, with some escaping cells, is consistent with the reduced number of normal sized adipocytes observed in adipose tissues from FIGIRKO mice and is more than sufficient to produce a strong whole body phenotype.
The phenotype of the FIGIRKO mice differs drastically from that of mice with a fat specific knockout of the insulin receptor (FIRKO). While both mice have reduced white fat mass, this change is much greater in FIGIRKO mice where there is a decrease in cell number compared to FIRKO mice where there is primarily a change in cell size. Both FIRKO and FIGIRKO mice are protected against age-associated glucose intolerance and diet-induced obesity and glucose intolerance, and both display increased energy expenditure 14, 25
. On the other hand, FIGIRKO mice are extremely cold sensitive and unable to exhibit an adequate thermogenic response when placed in the cold, whereas FIRKO mice show normal temperature regulation, indicating that the combined inactivation of the IR and IGF1R mediates some additional effects from that of IR alone on BAT development and function.
These additional effects of the IGF1R are only observed when combined with a deletion of the IR. Thus, mice with adipocyte specific deletion of IGF1R only have normal BAT mass, normal temperature regulation and even an increase in white adipose tissue mass. The latter is part of a generalized increase in somatic growth, which has been attributed to increased circulating IGF-1 in these mice 15
. However, FIGIRKO mice have normal IGF-1 levels. The reduction in both white and brown adipocyte numbers in FIGIRKO mice reflects the important and synergistic roles of insulin and IGF-1 on the control of white and brown adipocyte differentiation.
Both white and brown adipose tissues from FIGIRKO mice show reductions in adipocyte differentiation markers. Furthermore, IR and IGF1R double knockout brown preadipocytes (DKO) fail to accumulate lipids or increase expression of adipocyte markers after induction of differentiation. C/EBPβ phosphorylation was strongly reduced in DKO cells, both under basal conditions or after induction of differentiation, indicating that insulin and IGF-1 signaling play a critical role in the control of adipocyte differentiation via activation and phosphorylation of C/EBPβ leading to impaired induction of C/EBPα and PPARγ.
Interestingly, while insulin and IGF-1 signaling are essential for adipose development in the preformed, discrete brown adipose tissue depots, they may not be required for differentiation of systemic brown adipocytes. These differences may be due to the differential lineages of these different brown fat depots 22, 23
. This is also suggested by different molecular responses of precursors from these depots to a stimulation by a PPARγ agonist 26
. It is also possible that this pool of brown preadipocytes has a low level of aP2 expression compared to the preformed depot, limiting the extent of receptor inactivation in this population of cells.
FIGIRKO mice are protected against age- and HFD-induced glucose intolerance. Interestingly, expression of inflammatory markers in adipose tissue and circulating cytokine levels are increased in FIGIRKO mice on high fat diet to a similar level as in control mice, ruling out reduced inflammation in fat as a cause for the improved glucose tolerance observed in these mice. Improved glucose tolerance is also not due to differences in insulin secretion. However, insulin-stimulated glucose uptake is increased in skeletal muscle from FIGIRKO mice, and coupled with the increased insulin levels in HFD-fed FIGIRKO mice, it seems likely that the better glucose tolerance of FIGIRKO mice is due to increased insulin action in skeletal muscle.
The most dramatic finding in FIGIRKO mice is the almost complete absence of interscapular BAT, highlighting the crucial role for insulin/IGF-1 signaling in BAT formation. BAT mass/activity in humans is inversely correlated with body mass index and percent body fat 2–4, 27–29
. Interestingly, the defect in brown fat development in FIGIRKO mice is associated with increased energy expenditure and leanness. Although surprising, this is in agreement with the phenotype observed in UCP1 deficient mice which are cold sensitive30
and protected from HFD-induced obesity when raised at 20°C 31
, but can become obese on HFD in a thermoneutral (29°C) environment32
. This suggests that for BAT to have a role in regulating energy expenditure, the tissue must have functional insulin and IGF-1 receptors. Exactly how FIGIRKO mice maintain temperature at 21°C is unclear, since FIGIRKO mice do not show increased systemic brown adipocytes or increased expression of any UCP in muscle, fat or liver. Nevertheless, at 4°C FIGIRKO mice are unable to maintain their body temperature, indicating that brown fat is essential for adaptative thermogenesis, similar to what has been observed in mice with UCP1 inactivation 30
. However, UCP1 deficient mice can be kept at 4°C if they are gradually adapted to the cold 33, 34
, but this is due to shivering rather than the development of BAT-related thermogenesis 33, 35
Cold induced thermogenesis is mediated via the sympathetic nervous system through activation of β-adrenergic receptors 36, 37
. Despite the small BAT mass, the increase in FDG uptake after β3 stimulation is greater in FIGIRKO mice than controls. Furthermore, the β3 adrenergic agonist induced UCP1, PGC1α and Elovl3 expression in white and brown adipose tissues from both control and FIGIRKO mice to the same extent. However, the relative amount of FDG uptake is still lower in BAT from FIGIRKO mice in both basal and stimulated conditions indicating that in addition to the reduced BAT mass, BAT from FIGIRKO mice is less active.
Taken together, these results highlight the critical role of insulin and IGF-1 signaling in the control of white and brown adipose tissue development and function, as well as the regulation of glucose metabolism and energy expenditure.