Spinal cord injury (SCI) causes partial or total interruption of neural signal transmission between the brain and the periphery, thereby limiting physical activity. Chronic SCI, due to an extremely sedentary lifestyle, invariably results in adverse body composition changes characterized by decreased lean mass and increased adiposity. Such changes are associated with metabolic dysfunction (1–5) leading to an increased risk for cardiovascular disease, liver disorders, and type 2 diabetes (T2DM) compared to the general population (6–9). However, the underlying mechanisms for the predisposition of persons with SCI to metabolic perturbations and the associated cardiac and hepatic disorders remain unclear. Several lines of evidence suggest that alterations in the liver or visceral adipose tissue including neutrophil infiltration, macrophage activation, increased expression of pro-inflammatory cytokines and hepatic lipid accumulation (10–13) which often is associated with development of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) (14, 15). Furthermore, persons with SCI have decreased anabolic hormone levels (16) which are associated with abnormal lipid and metabolic profiles. A clear understanding of hepatic pathology that develops after SCI may provide new insight into the development of impairments of fat and carbohydrate metabolism. However, limited studies have addressed the details of pathophysiological events which occur in the liver after SCI. The mechanism underlying dyslipidemia and whether this change precedes, or is the cause of, systemic metabolic dysfunction after SCI is not known. In addition, the potential mediators, if any, that regulate the interaction between hepatic function and other tissues to achieve systemic metabolic homeostasis after SCI have yet to be investigated.

Fibroblast growth factor-21 (FGF21) is a metabolically active peptide primarily secreted by the liver in response to various nutritional, physiological and pathological stimuli (17). FGF21 is an integral component of the hormone network that modulates the action of insulin, leptin, glucagon-like peptide (GLP1), adiponectin, growth hormone, and insulin-like growth factor (18). FGF21 has been shown to regulate carbohydrate and lipid metabolism in the liver, adipose tissue, skeletal muscle, pancreas, and central nervous system (18–20). In animal models, FGF21 corrects multiple abnormalities of metabolism, including those present in persons with insulin resistance and T2DM (21–23). FGF21 reduces levels of serum triglycerides (TG), total cholesterol, and low-density lipoprotein (LDL)-cholesterol but raises the level of high-density lipoprotein (HDL)-cholesterol, all of which changes represent favorable alterations to the lipid profile (19, 21). Transgenic mice overexpressing FGF21 exhibit resistance to the development of high fat diet (HFD)-induced obesity (24). In addition, FGF21 exerts hepato-protective effects by promoting hepatic lipid and free fatty acid (FFA) metabolism, reducing hepatic adiposity, and ameliorating NAFLD and NASH (19, 22, 25–27). Moreover, FGF21 can act on cardiac muscle to protect against heart disease (28) and ameliorates obesity-related inflammation in a rat model reducing inflammatory cytokine production (29). The molecular mechanisms by which FGF21 produces its physiological effects are, however, still being elucidated. FGF21 transcription is induced by peroxisome proliferator-activated receptor (PPAR)-γ and PPAR-α, which are the targets of agents prescribed to treat T2DM (thiazolidinediones) and hyperlipidemia (fibrates), respectively (23, 30).

FGF21 requires the obligate coreceptor β-klotho (KLB) for cellular signaling both in vivo and in vitro (31). The binding of FGF21 to KLB enables its interaction with the FGF receptor 1 (FGFR1), thus activating intracellular signaling pathways (32). FGF21 signaling is tissue-specific and is chiefly determined by tissue distribution of KLB. Adipose tissue has high levels of KLB expression and is a primary site of FGF21 action (32, 33). In obese individuals, serum FGF21 levels positively correlate with the subcutaneous adipose tissue (SAT) area (34). FGF21 knockout mice show less SAT and are more insulin-resistant when fed a HFD (24, 34). Mechanistically, FGF21 improves systemic insulin sensitivity by promoting the healthy expansion of SAT (34), thereby increasing serum levels of adiponectin, a major adipokine produced by adipose tissue, which mediates the beneficial effects of FGF21 on the liver and muscle (35, 36). Adiponectin signaling is mediated by activation of two types of receptor: type 1 (AdipoR1) is predominantly expressed in skeletal muscle and activates AMP-kinase; the type 2 adiponectin receptor (AdipoR2) is predominantly expressed in the liver and activates the PPARα signaling pathway (37). Of note, the magnitude of FGF21-induced weight loss is reduced in leptin-deficient mice (33), implying a possible requirement of a functional leptin axis for FGF21 action on body weight regulation (38). While the beneficial effects of FGF21 on metabolic homeostasis have been extensively studied in able-bodied individuals, FGF21 expression and function after SCI has not been investigated.

Persons with SCI have been reported to have reduced energy expenditure resulting from a marked reduction in lean tissue mass and in the ability to ambulate and/or exercise (39, 40). On the other hand, recent reports indicated increased total food intake in persons with SCI when compared to individuals who were immobilized for reasons other than SCI (41, 42). In addition, analysis of the pattern of food intake of overweight or obese persons with SCI suggests that there is an increased proportion of total calories consumed from fat (43). These post-SCI dietary changes almost certainly accelerate the accumulation of fat in various body compartments. However, studies that investigated nutritional status in chronic SCI are limited, and knowledge with respect to how macronutrients impact metabolic gene expression and dysregulation of metabolic homeostasis after SCI is lacking. Furthermore, the relationship between serum FGF21 levels and SCI is unknown.

Using a spinal cord transection model, a high fat diet was recently reported to induce glucose intolerance without increasing fat mass or liver weights (44). To understand the possible role of FGF21 in altered fat, carbohydrate and energy metabolism following SCI, blood and tissue samples collected from SCI mice fed either a high fat diet (HFD) or control diet (ConD) were used to determine circulating FGF21 levels and expression of FGF21 mRNA. The cause of the potential perturbations in FGF21 levels and, if altered, the probable impact on lipid and carbohydrate metabolism was investigated by analyses of relevant signaling and molecular changes in fat, liver and muscle.

The central question addressed in this study was whether SCI per se has an impact to change serum FGF21 levels or activity of FGF21-dependent signaling, including that through adiponectin, in a mouse model of complete spinal cord transection. To better understand the meaning of changes in levels of these regulatory factors, the analysis employed a broad approach that examined signaling in liver, iFAT, oFAT and skeletal muscle which, collectively, are responsible for most effects of insulin on blood glucose levels, with adipose tissue and liver also playing key roles in synthesis, storage and release of fats. The principal conclusions of our work herein are that SCI reduced serum FGF21 and adiponectin levels, including total and the more bioactive HMW moiety, and the expression of hepatic FGF21 and adipocyte expression of adiponectin. The data suggest that SCI reduced signaling in adipocytes in response to FGF21 via KLB and FGFR1, and diminished adiponectin action on the liver through reduced hepatic AdipoR2 expression and decreased PPARα activation. In addition, SCI increased levels in liver of inflammatory cytokines, including IL-1β, IL-6 and CD11b, as well as in both iFAT and oFAT of TNFα mRNA, the changes were exacerbated by HFD suggesting that these adipose tissue deposits contribute to the state of low-grade chronic inflammation reported after SCI (58), which was associated with elevated liver expression of ALT mRNA and serum levels of ALT, indicative of hepatocellular injury. Moreover, SCI reduced insulin receptor in gastrocnemius muscle, and reduced mRNA levels for IRS-1 and Glut-4 in iFAT and oFAT, and IRS-1 in liver suggesting impaired insulin signaling pathway after SCI. Evidence of perturbations in regulation of synthesis of steroids, fatty acids and fatty acid metabolism after SCI included greatly elevated HMGCR mRNA expression and ACCα activity in liver after SCI. During administration of a HFD, serum FFA rose in SCI mice on HFD, but not control mice, suggesting impaired ability to store FFA as triglycerides in adipocytes or to use FFA as fuel in muscle and other tissues. FABP4 mRNA was elevated in liver of SCI-HFD mice which is notable because serum levels of the corresponding protein are linked to T2DM and other disorders (52). Taken together, the data indicate defects in metabolism of fats and carbohydrates in multiple tissues after SCI and implicate profound dysregulation of the FGF21 – adiponectin metabolic regulatory circuit as one key driver of these many perturbations.

Hepatic FGF21 expression is regulated by many factors that include nutrients, physical activity and medications (17, 59). Reasons that hepatic FGF21 expression is reduced by SCI are unclear. Because of the key role played by PPARα in activation of FGF21 transcription in liver, the reduced nuclear levels and transcriptional activity of PPARα provide insight with respect to one possible mechanism by which FGF21 mRNA levels are reduced in liver after SCI (60), but other possibilities exist. While it remains uncertain how exercise increases FGF21, the linkage between physical activity and FGF21 suggests that just as exercise raises levels of this hepatokine (61, 62), a sedentary lifestyle may lower them. The possibility should also be considered that cues from hepatic or adipocyte inflammation, which was present in both adipose tissues (iFAT and oFAT) and liver, as suggested by the elevated expression of mRNA levels for inflammatory cytokines, particularly for TNFα in the adipose tissue, and IL-1β, IL-6 and CD11b mRNA in the liver, could lead to reduced hepatic FGF21 expression. These data suggest that an increased focus on derangements in liver function as a key factor in impaired fat and carbohydrate metabolism after SCI should be considered.

The reduction in adipocyte expression of adiponectin mRNA and serum adiponectin level observed in spinal cord transected mice in the current study may be linked in part to the lower level of serum FGF21 observed after SCI; the data also suggest resistance to FGF21 actions based on lower expression of FGFR1 and KLB in iFAT and oFAT cells. While controversial, some evidence for FGF21 resistance has been presented (63). Other explanations cannot be excluded from the data available and include reduced capacity for PPARγ to activate adiponectin transcription as a result of local pro-inflammatory signals (49). Whatever the mechanisms responsible for reduced serum adiponectin after SCI might be, they were coupled with several molecular changes in liver that might be interpreted as indirect evidence of resistance to adiponectin action. These include lower levels of AdipoR2 mRNA and PPARα activity. While not extensively studied, the concept of adiponectin resistance has been proposed previously and some experimental support for its existence has been presented (64).

Lower expression in iFAT and oFAT of leptin and reduced serum leptin levels were also observed. These changes are consistent with a prior report of low serum leptin after a contusion SCI in rats (65). Implications of these changes are unclear but it should be noted that leptin has been implicated in multiple biological responses including of FGF21 action (33, 38). Regulation of leptin expression remains incompletely understood, but it is clear that leptin levels correspond to adipose tissue mass and are reduced by low calorie intake (66). Thus, the low mass of adipose tissue deposits in the SCI mice (44) observed in this investigation may explain, at least in partial, the low leptin levels observed.

Serum FFA levels were significantly elevated in HFD-fed SCI mice compared to SCI-ConD mice but not in sham-HFD mice. These observations suggest impaired ability of SCI mice to clear FFA from the circulation or to metabolize FFA such that they accumulate in blood. Elevated serum FFA levels have been shown to reduce tissue insulin sensitivity through multiple mechanisms that include reduced glucose uptake by skeletal muscle and increased TNFα release by liver Kupfer cells (67, 68), Importantly, elevated FFA levels are a risk factor for insulin resistance that may be independent of total fat mass (56). Elevations in serum FFA in SCI-HFD mice were associated with increased hepatic expression of FABP4. FABP4 is thought to have numerous functions that include its ability to bind free fatty acids and retinoids within cells, to participate in uptake of FFA from the extracellular space, and to route FFA to nuclear PPAR receptors to appropriately regulate the gene expression programs they control (69, 70); the implications of elevated hepatic FABP4 following SCI fed with a HFD are uncertain, but it is noteworthy that elevated serum levels of the protein encoded by this gene showed a positive correlation with high-sensitivity C-reactive protein (a measure of systemic inflammation), lipid parameters, and the homeostatic model assessment of insulin resistance (HOMA-IR) (56, 69–71). Taken together, these findings indicate that following SCI, mice were less tolerant of a HFD, and that this type of diet elicited several significant and deleterious changes.

Effects of SCI on the serum lipid profile have been evaluated in several other studies. Elevated serum triglycerides were observed at one month after SCI in T3-transected rats associated with increased visceral adiposity (72). In a separate study, serum FFA and glucose were elevated 42 days after a 200 kdyne contusion injury in rats (73). While extensive study of serum lipid profiles has been performed in patients with SCI (1), to date these studies have not characterized serum FFA levels.

An unexpected finding was the upregulation in SCI-ConD mice of HMGCR, a rate-limiting enzyme in the synthesis of cholesterol, possibly reflecting increased tissue demand, which might be expected during reparative responses in the spinal cord or to support neuroplasticity. In SCI patients, abnormalities of lipid metabolism include reduced HDL cholesterol (1, 2) and elevated triglycerides in response to a high fat challenge when compared to data for historical controls (74). In rats that were studied 1 month after thoracic level-10 transection, serum triglycerides, total and HDL cholesterol levels tended to be lower as compared to sham-operated controls, although this difference was not significant (72). Further study is needed to understand how SCI impacts metabolism of fats or how SCI perturbs the networks of adipokines and hepatokines that regulate them.

The data presented suggest that at 84 days after spinal transection, there is accumulation of tissue macrophages in the liver, as reflected by increased expression of inflammatory cytokines, including IL-1β, IL-6 and CD11b. There is also some evidence of inflammation in fatty tissue based on elevated expression of TNFα mRNA in both iFAT and oFAT, and increased IL-1β mRNA in oFAT. These alterations would be expected to stress hepatocytes, impair insulin action on hepatocellular regulation of glucose uptake and glucose output and, possibly, injure hepatocytes as well. Evidence of hepatocellular impairment, and possibly injury, was observed in post-SCI mice based on elevated circulating levels of ALT, as well as increased hepatic ALT expression. It is noteworthy that Kupffer cells, the resident hepatic macrophages, have been linked to NAFLD, a risk factor for insulin resistance (75), thus indirectly implicating the changes in CD11b to liver disease and insulin resistance after SCI. Elevated cytokine expression in fat deposits is also thought to be a risk factor for impaired glucose homeostasis (76, 77), suggesting that such alterations in adipose tissues observed in the current study may further contribute to impaired glucose handling and insulin action in SCI mice. This postulate is indirectly supported by findings of reduced expression of several key genes in insulin signaling in iFAT, oFAT, liver and gastrocnemius muscle. However, the data do not establish causal relationship between TNFα expression in adipose tissue and altered fat or carbohydrate metabolism. Also unclear is the cellular source of cytokines in adipose tissues, but prior studies indicate that infiltration of fat deposits by tissue macrophages is the primary explanation for such changes (76). Evidence of hepatic inflammation and hepatocellular injury that increases with time to 21 days after SCI has been observed in rats following a 200 kdyne contusion SCI. In the same study, elevations in serum ALT were observed in rats with cervical (C5), thoracic (T8) and lumbar (T12) contusion injuries (11). In that study, hepatic mRNA levels for IL-1ß increased at days 3 and 7 post-SCI while TNFα increased at 14 and 21 days, suggesting a time-dependent response. Hepatic lipids, assessed by Oil red O staining, were also increased at 14 days post injury (11). A more recent study from the same group found elevated hepatic CD11b at 6-weeks after a 200 kdyne injury at T8 that was associated with increased mRNA levels for several cytokines, including TNFα and IL-1ß, and increased hepatocellular lipids and iron levels (73). These findings are in agreement with the increased expression of CD11b in liver in the current study. However, no significant alteration in hepatic expression of cytokines was observed in the present study. Reasons for these discrepant findings are unclear but may include differences in time since injury, species and/or responses to contusion versus transection SCI.

It was previously reported that, in mice, spinal cord transection results in marked decreases in weights of iFAT and oFAT and that HFD did not increase weights of these fat deposits in SCI mice (44). Similarly, a reduction in total fat mass in rats after transection at thoracic level-4 has been reported and was associated with a small increase in metabolic rate and food intake (78). There is insufficient data to determine why the observed decreases in fat depots occurred. It has been argued that because body weights of the mice from which tissues were obtained for the current study increased over time after SCI that their net caloric intake exceeded metabolic demands. However, it remains unclear why gains in body weight were not associated with a return toward normal adipose tissue mass; possible explanations include impaired ability of adipocytes to take up and store lipids or, possibly, a loss of the normal replacement of aging adipocytes from the pool of adipocyte progenitors such that adipose tissues become depleted of healthy and fully functional adipocytes. Similarly, the reduced levels of PPARγ and ASC-1 observed in the current study could simply reflect low fat mass. Adipocytes undergo turnover whereby there is constant replenishment of aging or damaged adipocytes ones formed from a committed pool of progenitors (79). Thus, failure of HFD to promote gains in adipose tissue mass may indicate damage to adipocytes and/or inadequate replenishment of adipocytes from pools of progenitors. Of note, it was reported that FGF21 knockout in mice displayed disorders of adipose tissue due to defects in PPARγ signaling and PPARγ-dependent gene expression (34, 80). This report demonstrates that it is plausible that the reduced levels of FGFR1 and PPARγ activity observed in iFAT and oFAT could result in dysfunction of adipose tissue, low adipose tissue mass and impaired adiponectin expression and release.

It is now accepted that fat deposits are highly specialized in their functions depending on their location. oFAT is particularly important when considering the pathogenesis of insulin resistance and diabetes, perhaps because adipokines and free fatty acids liberated by oFAT are sent to the liver by the portal circulation. Unexpectedly then, in the present study, gene expression changes in iFAT and oFAT from SCI mice, and responses in SCI mice of these fat deposits to HFD were similar in magnitude and direction suggesting that physiological adaptations to SCI in mice overcame phenotypic differences related to localization of these fat deposits.

It was previously reported that HFD induced glucose intolerance in spinal cord transected mice at 84 days after injury and that fasting glucose was modestly increased in SCI-ConD mice at this time point (44). When these findings are taken together with the ability of FGF21 to improve insulin action, and low FGF21 observed in SCI in mice, it was of interest to examine insulin signaling pathways in tissues from these SCI mice. Whereas no reduction in IRS-1 or Glut-4 mRNA was observed in skeletal muscle, reduced levels of IRS-1 mRNA were observed in iFAT and oFAT, as well as liver, and these changes were not influenced by administration of a HFD. IRS-1 is critical to coupling insulin receptor and downstream signaling pathways while Glut4 represents the primary mechanism for insulin-induced glucose uptake. The findings suggest that, in the spinal cord transected mouse model at least, loss of insulin responsiveness of liver and fat deposits is an important mechanism leading to insulin resistance and diabetes. However, as discussed above, the data do not exclude a possible deterioration of function of adipose tissue as an explanation for the uniform decrease in insulin signaling pathways genes in adipose tissues. Further studies are necessary to better understand the respective roles of fat, liver and muscle in development of T2DM after SCI.

A limitation to be considered when interpreting our findings or considering their translation is that the approach employed did not directly test for physiological significance of lower FGF21 levels in the SCI mouse model used. Consequently, although it is attractive to posit that the changes observed downstream of serum FGF21 may be attributable to the lower serum FGF21 levels observed after spinal cord transection, this mechanistic linkage is yet to be proven. It should also be noted that whereas persons with SCI usually gain body fat mass, mice with spinal cord transection lost fat mass (44). This difference may limit how well changes in the spinal cord transection mouse model used in the current model reflect what occurs in individuals with SCI. Another limitation of these studies is that they evaluated only male mice; males were selected because male C57B6 mice are more sensitive to high-fat-diet-induced glucose intolerance than females. We expect that the pattern of changes in serum and hepatic FGF21 levels would be the same between males and females after SCI although some gender effects on the magnitude of changes cannot be excluded.

In summary, this study, the impact of SCI on expression of and signaling by FGF21 was investigated. Major findings of the study are that at 84 days after a complete spinal cord transection, hepatic FGF21 expression and serum FGF21 protein were reduced, associated with lower serum total and HMW adiponectin and leptin. A HFD raised serum FFA in SCI but not sham mice suggesting impaired ability for tissue to take up or metabolize fats. Prior studies demonstrated glucose intolerance in the SCI-HFD mice model that was used for these studies. Decreased expression for insulin signaling-related genes were observed in adipose tissue and liver but, surprisingly, not skeletal muscle. Taken together, reduced levels of FGF21 and adiponectin provide attractive mechanisms for the links between SCI and insulin resistance and T2DM. Moreover, the findings suggest key roles for perturbed function of adipose tissue and liver in the pathogenesis of T2DM after SCI. Further research will be needed to formally prove a causal relationship between abnormal levels of these proteins and carbohydrate and fat metabolism after SCI.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Institutional Animal Care and Use Committee at the James J. Peters VA Medical Center.

X-HL designed the study, carried out the research, analyzed, interpreted the results, and wrote the manuscript. ZG designed the study, carried out the research and edited the manuscript. LH carried out the research (animal studies). JP carried out the research. DA carried out the research. WB designed the study, analyzed, interpreted the results, edited the manuscript. JY analyzed, interpreted the results, edited the manuscript. CC designed the study, analyzed, interpreted the results, and wrote/edited the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by the Department of Veterans Affairs Office of Research and Development, Rehabilitation Research and Development (RR&D) Service. SPiRE 1I21RX001273-01 and PECASE B9280-O to JY, CDA-2 IK2RX002781-01 to ZG, National Center for the Medical Consequences of Spinal Cord Injury (B2020C), and by resources provided by the North Florida/South Georgia Veterans Health System and James J. Peters Veterans Affairs Medical Center.

The work reported herein does not represent the views of the US Department of Veterans Affairs or the US Government.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.