The present study confirms previous reports [21
] that IL6KO mice do not develop obesity or insulin resistance compared to wild-type controls. This is in contrast to the observations of Wallenius et al. [19
] and Matthews et al. [20
]. Although Wallenius et al. [19
] did not observe changes in weight or glucose tolerance before 6 months of age, the more recent study by Matthews et al. [20
] reported significant weight gain in conjunction with impaired glucose and insulin tolerance at 20 weeks old. While the reasons for the observed differences are unclear, the potential development of mature-onset obesity and metabolic changes could be affected by differences in dietary nutritional balance, regional background strain variation, and housing strategy. In fact, despite being only 2 weeks older than the mice used in the current study, the wild-type and IL-6KO mice used by Matthews et al. weighed approximately 10g and 15g more, respectively. This remarkable difference in body mass could have an important impact on metabolic parameters and, thus, experimental conclusions.
Absence of IL-6 did not improve systemic glucose and insulin tolerance in the genetically obese Lepdb
mouse model. Similar observations have been made in diet-induced obese (DIO) IL6KO mice [20
]. These reports indicate similar weight gain and fasted glucose levels, accompanied by modest elevation in blood glucose levels during a glucose tolerance test in DIO IL6KO mice compared to DIO IL6+ controls. In these models, it is feasible that in the absence of IL-6 other factors associated with diet-induced obesity continue to suppress insulin sensitivity [36
]. In contrast to these studies, Wunderlich et al. reported that hepatocyte-specific loss of IL-6 signaling impaired glucose and insulin tolerance in the basal state of lean animals, suggesting that IL-6 is required for physiologic maintenance of glucose homeostasis [37
]. The absence of a similar effect in our mouse model could reflect the difference between chronic, systemic absence of a signaling molecule and tissue-specific signaling inactivation. Additionally, a hyperinsulinemic-euglycemic clamp elicited a strong inflammatory response in the Wunderlich study [37
]. Under these conditions, absence of IL-6 signaling in hepatocytes reduced systemic insulin-stimulated glucose transport, indicating that IL-6 may preserve insulin sensitivity during acute inflammation. As a potentially important anti-inflammatory role of IL-6 was only observed during the clamp, it is unclear whether similar effects would be observed in the Lepdb
× IL-6KO mouse under the same conditions.
While we explored the contribution of IL-6 to the obese, insulin resistant phenotype of the Lepdb
mouse model, Sadagurski et al. [9
] examined the potential benefit of IL-6 over-expression on energy expenditure in the Lepob
mouse model. Although IL-6 over-expression dramatically protected against high-fat diet-induced obesity and insulin resistance, it was unable to fully prevent development of the obese, insulin resistant phenotype in the Lepob
mouse despite increased central leptin sensitivity. When combined with the currently reported results, the data indicate that chronic manipulation of IL-6 in the context of leptin deficiency or resistance does not alter development of obesity or systemic insulin resistance. Adding to the complexity, Chida et al. [21
] observed that combined IL-1 and IL-6 deficiency results in modest weight gain, while single knock-out controls remained lean. As IL-1 signaling has been implicated in central leptin action [38
], this result suggests a synergistic, central effect of these two molecules. The independent central effect(s) of IL-6 and the complicated cross-talk between leptin, IL-1, and IL-6, however, have yet to be fully elucidated.
The current study demonstrated that absence of IL-6 in Lepdb
mice leads to smaller increases in circulating glucose levels in response to a pyruvate bolus compared to IL-6+ controls. As the GTT and ITT did not indicate a systemic change in glucose utilization as a function of IL-6, the response to pyruvate is likely due to a reduction in hepatic utilization of pyruvate for the production of circulating glucose. IL-6 could control glucose output directly by altering glucose metabolism or indirectly through regulation of other metabolic pathways that require pyruvate as a substrate. Based on our observations, the effect of IL-6 deletion cannot be accounted for by changes in insulin receptor signaling or suppression of gluconeogenic gene expression since Pck
expression was decreased only in males. This does not completely rule out gluconeogenic control, however, as Samuel et al. [39
] observed increased endogenous glucose production via gluconeogenesis in diabetic rats and humans, independent of changes in Pck
expression. In light of this report it remains possible that allosteric regulation of fructose-1,6-bisphosphatase (FBPase) by F2,6P2 [40
] and subcellular localization of G6Pase [41
] are potential post-transcriptional regulatory targets for IL-6.
IL-6 has been reported to suppress insulin-mediated glycogen synthesis [3
] and directly stimulate hepatic glycogenolysis [43
]. Thus, removal of IL-6 could promote glycogen synthesis and/or reduce basal glycogenolysis, thereby reducing hepatic glucose output. Interestingly, Wunderlich et al. [37
] demonstrated that rendering hepatocytes unresponsive to IL-6 did not alter basal hepatic glucose metabolism or glycogen content, but increased hepatic glycogen synthesis during a hyperinsulinemic-euglycemic clamp. The former result is similar to our observation that basal glycogen content was similar in Lebdb
× IL6KO and IL6+ controls. A hyperinsulinemic-euglycemic clamp and radiolabelled glucose infusion may be required to more sensitively detect potential differences in glucose metabolism in our model.
In addition to direct effects on glucose metabolism, loss of IL-6 could enhance activity of other pyruvate consuming pathways at the expense of substrate availability for gluconeogenesis. We explored the possibility that Lepdb
× IL6KO mice display increased lipogenesis. Absence of IL-6 did not alter abundance of FAS or expression of SREBP-1c, the master regulator of lipogenesis [44
]. Additionally, basal hepatic triglyceride accumulation was similar in Lepdb
× IL6+ and Lepdb
× IL6KO mice. It remains possible that IL-6 could alter pyruvate flux by regulating pyruvate dehydrogenase activity, altering lactic acid formation, or modulating cellular respiration/mitochondrial consumption of pyruvate in Lepdb
mice. Interestingly, Matthews et al. [20
] observed altered hepatic mitochondrial function in DIO IL6KO mice in association with hepatic inflammatory infiltrates and reduced insulin-stimulated Akt activation. Given that pyruvate carboxylase is localized to the mitochondria and required for the utilization of pyruvate in gluconeogenesis [45
], it could be hypothesized that altered hepatic mitochondrial function in the Lepdb
× IL6KO model would result in decreased glucose production during the pyruvate tolerance test.
Absence of systemic IL-6 in female Lepdb
mice modestly reduced markers of hepatic inflammation, including transcription of Saa1
. Given that low-grade activation of the acute phase response is associated with increased hepatic glucose output [46
], absence of IL-6 may blunt activation of the acute phase response and subsequently reduce hepatic usage of pyruvate for glucose production. Inhibition of NFκB-mediated inflammation has also been associated with restored suppression of hepatic gluconeogenesis and reduced glucose production from pyruvate in Lepdb
]. A modest reduction in expression of Rela
in female Lepdb
× IL6KO mice is consistent with this latter effect.
In summary, absence of IL-6 in Lepdb mice improved pyruvate tolerance in association with modest reduction in hepatic inflammation, but no apparent improvement in insulin receptor signaling. This study provides further support for a contributory role of IL-6 to metabolic dysregulation in the Lepdb mouse model, but further studies will be required to define the precise mechanism of IL-6 action.