Obesity-mediated insulin resistance has been shown to have a strong inflammatory component. Several groups, including Weisberg et al. [11
] and Xu et al. [9
], have suggested an important role for the adipose tissue-associated macrophage in mediating this inflammatory state and subsequent insulin resistance. In contrast to the characterization of the adipose tissue-associated macrophages, the effect of obesity on the Kupffer cell population is poorly understood. The current study investigated the role of liver macrophages, or Kupffer cells, in a diet-induced obese mouse model of hepatic inflammation and insulin resistance. Our results support the conclusion that Kupffer cells do not contribute to the proinflammatory environment of obesity, but modulate STAT3-dependent signaling and obesity-associated impairment of hepatic lipid metabolism and insulin resistance.
Here we report similar levels of macrophage markers, Emr1
, in livers of lean and obese mice, suggesting no expansion of the Kupffer cell population in obesity. F4/80 staining of liver samples from lean and DIO mice supports this interpretation. This is also supported by the observations of Weisberg et al. [11
] and Xu et al. [9
] who reported similar Kupffer cell numbers despite an increased adipose tissue-associated macrophage population. Cintra et al. [40
] and Cai et al. [41
], however, report increased Kupffer cell numbers in mice placed on a high-fat diet for 2 and 3 months, respectively. The use of the Swiss mouse strain by Cintra et al. [40
] could account for some of this difference, as mouse strains display varied phenotypes. A variation in diet composition, such as the ratio of saturated to unsaturated fat [42
], could play a role in the obesity-associated effects seen by Cai et al. [41
], but this dietary information was not provided.
In addition to no change in Kupffer cell markers in the liver following high-fat feeding, the absence of a change in hepatic inflammatory cytokine expression suggests that Kupffer cells are not actively contributing to the proinflammatory environment of diet-induced obesity. A collective increase in expression of alternative activation markers suggests that hepatic macrophages do not respond to the Th1 polarizing environment of obesity. These data are in contrast to adipose tissue-associated macrophages which undergo a Th2 to Th1 switch [13
] and actively increase production of inflammatory cytokines. Other hepatic immune populations like CD4+
NK1.1 cells can mediate alternative activation of Kupffer cells in response to inflammation [43
]. Perhaps they also mediate a similar Kupffer cell activation in response to the adipose tissue-derived cytokines and hepatic lipid accumulation.
Kupffer cell-ablated and IL-10KO DIO mice displayed increased hepatic STAT3-dependent signaling. In all cases, this occurred in the absence of increased hepatic IL-6 message, but a dramatic (≥90%) reduction of IL-10. Since IL-10 is an antagonist of IL-6 signaling, we hypothesize that Kupffer cell-derived IL-10 could modulate this response. This observation does not rule out a role for other Kupffer cell-derived direct or indirect cellular mediators of hepatocyte STAT3 signaling. Kupffer cell-derived molecules could be inducing inhibitors of IL-6 or other STAT3-dependent effectors within the hepatocyte, such as protein inhibitor of activated STAT3 (PIAS3), SH2-domain-containing tyrosine phosphatase (SHP2), or p38 stress kinase [44
]. Alternatively, increased shedding or expression of soluble IL-6 receptor alpha (soluble gp80) following Kupffer cell ablation, could sensitize hepatocytes to circulating IL-6 and increase acute phase protein production [47
]. Endotoxin stimulation of CD14 has been reported to activate the acute phase response, especially in the obese state [49
], but this seems unlikely in our models as NFκB transcriptional markers remained unaltered.
Both loss of Kupffer cells and systemic absence of IL-10 exacerbated hepatic triglyceride accumulation (steatosis) associated with obesity. This suggests that Kupffer cells and/or IL-10 production impart partial protection against pathologic accumulation of hepatic lipid. In support of this premise, inhibition of Kupffer cells has been shown to decrease PGE2 release within the liver leading to increased hepatic lipid synthesis [50
]. In agreement with our DIO IL-10KO model, den Boer and associates also observed that systemic loss of IL-10 during obesity results in hepatic triglyceride accumulation [51
]. Loss of IL-10 and increased hepatic STAT3-dependent signaling in our model is not sufficient, however, for increased hepatic lipid accumulation, as lipid content remained unaltered following Kupffer cell ablation in lean mice. A high-fat diet appears to also be necessary. These observations do not rule out the possibility that other Kupffer cell-derived factors could be directly involved in hepatic triglyceride synthesis or secretion during obesity.
Experimental evidence demonstrates that chronic inflammation and altered lipid metabolism associated with obesity directly impair insulin signaling. While many cytokines are involved in the chronic inflammation of obesity, IL-6 appears to be a major effector inhibiting insulin signaling in the liver at least in part through SOCS-3 induction [5
]. Lipid accumulation induces oxidative stress and activates serine kinases that impair the insulin-signaling cascade [54
]. Our Kupffer cell-ablated DIO mice displayed significant increases STAT3-dependent signaling and hepatic triglyceride accumulation. As a result, we hypothesized that insulin signaling would be impaired in this model. Hepatic response to an insulin bolus following Kupffer cell ablation was impaired 30-40% in association with a 3-fold increase in expression of Socs3
in the DIO mice. Despite an increase in HOMA-IR, systemic response to insulin, glucose, or pyruvate was not significantly altered following ablation in DIO mice. These data would suggest that Kupffer cells promote local hepatic insulin action. Since Kupffer cell ablation in lean mice elevated STAT3-dependent gene expression in the absence of impaired insulin signaling, increased STAT3-dependent signaling alone is not sufficient to impair insulin signaling in this model.
Recently, Cintra et al. [40
] observed that treatment of DIO Swiss mice with IL-10 neutralizing antibodies or suppressing its expression using antisense techniques increased inflammation and impaired insulin responsiveness. In support of this premise, increased IL-10 expression levels in humans have been correlated with increased insulin sensitivity [57
]. While infusion of IL-6 into mice impaired insulin responsiveness during a hyperinsulinemic, euglycemic clamp study, co-infusion of IL-10 was shown to overcome the IL-6 effect [58
]. In contrast to this, insulin signaling remained unaltered in our DIO IL-10KO mouse model. This agrees with the observation by den Boer et al. [51
] that DIO IL-10 knockout mice displayed altered hepatic lipid metabolism, but unaltered insulin action compared to high-fat-fed wild-type controls. Additional work is needed to clarify the relationship between IL-10, inflammation, and insulin signaling.
Our observations indicate that while IL-10 is associated with modulation of hepatic STAT3-dependent signaling and lipid metabolism in DIO mice, one or more additional Kupffer cell mediators is necessary to partially preserve insulin responsiveness. Reactive oxygen species and ER stress have been directly implicated in the induction of insulin resistance in hepatocytes [55
]. Kupffer cell-produced anti-oxidative molecules are potential direct or indirect protective mediators. Activation of the powerful anti-oxidant heme oxygenase-1 (HO-1) [60
] reduces obesity-associated inflammation and improves systemic insulin responsiveness in mouse and rat models [61
]. Kupffer cells produce HO-1, which has been demonstrated to protect against endotoxemia and oxidative stress in a rat model of ischemia-reperfusion [63
]. Kupffer cell ablation may alter synthesis of another powerful anti-oxidant, lipoic acid, which can dampen macrophage inflammatory responses [64
] and has also been implicated in protection against oxidative stress-induced insulin resistance [65
]. Cyclooxygenase (COX) 1/2 and prostaglandin E2 (PGE2) appear to be integral to resolution of an inflammatory response [67
] and lipid accumulation [50
]. Both of these latter responses appear to be required for impairment of insulin signaling in our DIO Kupffer cell ablation model.
Very recently, Neyrinck et al [68
] demonstrated that inhibition of Kupffer cells in DIO mice by i.p.
injection of gadolinium chloride improves HOMA-IR and systemic glucose tolerance in association with reduced hepatic steatosis. While these results are contrary to those reported here, there are important limitations to the gadolinium chloride model that may account for this difference. Gadolinium chloride treatments are associated with hepatocyte proliferation [69
]. Gadolinium chloride toxicity and mineral deposition in liver and stomach of rodents has also been documented by Spencer et al. [70
] following single injections. The markedly reduced weight gain on a high fat diet observed by Neyrinck et al [68
] in mice receiving twice weekly gadolinium chloride injections for 3 weeks may be a related to these tissue changes. Improved insulin sensitivity and glucose tolerance in gadolinium chloride-treated DIO mice could be due to the decreased weight gain independent of changes in Kupffer cell function.
In summary, the results of this investigation indicate that Kupffer cells resist classical activation and do not contribute to the proinflammatory state of high-fat diet-induced obesity. Loss of Kupffer cells in obesity results in a marked loss of hepatic IL-10 expression that is associated with increased hepatic STAT3-dependent signaling and increased lipid accumulation. Kupffer cell loss in DIO mice is also associated with decreased hepatic insulin receptor signaling. Future investigations are necessary to elucidate the mechanism of these Kupffer cell-dependent effects.