Pro-inflammatory cytokines of a T helper-1-signature are known to promote insulin resistance (IR) in obesity, but the physiological role of this mechanism is unclear. It is also unknown whether and how viral infection induces loss of glycemic control in subjects at risk for developing diabetes mellitus type 2 (DM2). We have found in mice and humans that viral infection caused short-term systemic IR. Virally-induced interferon-γ (IFN-γ) directly targeted skeletal muscle to downregulate the insulin receptor but did not cause loss of glycemic control because of a compensatory increase of insulin production. Hyperinsulinemia enhanced antiviral immunity through direct stimulation of CD8 + effector T cell function. In pre-diabetic mice with hepatic IR caused by diet-induced obesity, infection resulted in loss of glycemic control. Thus, upon pathogen encounter, the immune system transiently reduces insulin sensitivity of skeletal muscle to induce hyperinsulinemia and promote antiviral immunity, which derails to glucose intolerance in pre-diabetic obese subjects.

Here, we investigated how infection impacts insulin-mediated regulation of glycemia. We found that virally-induced IFN-γ caused IR in the skeletal muscle through direct engagement of the IFN-γ receptor on myocytes and down-modulation of the insulin receptor on these cells. Infection-induced selective IR drove an increase of systemic insulin concentrations to prevent hyperglycemia, but also to boost the anti-viral CD8 + T cell response through direct promotion of effector cell function. When systemic insulin sensitivity is already reduced, such as in pre-diabetic obese subjects due to hepatic IR, infection overwhelmed the ability of the endocrine system to compensate for increased muscle IR and glucose intolerance (GI) ensued. We thus identify an immune-endocrine regulatory feed-back mechanism of antiviral immunity and provide additional insights in the underlying physiology of DM2.

It is currently unclear what the physiological role is of reduced systemic insulin sensitivity following infection. Immune activation comes at a considerable energetic cost () as cells switch from oxidative to glycolytic metabolism (). It was therefore proposed that inflammation-induced IR is a physiological response to infection that aims to increase systemic glucose availability to activated immune cells (). However, infection-induced acute loss of glycemic control is only observed under extreme conditions such as sepsis, whereas DM2 is associated only with low amounts of systemic inflammation (). Alternatively, inflammation-induced IR may be a strategy of the immune system to involve endocrine mediators in the response against infection. Both cytokines and hormones regulate the metabolism of cells in response to alterations in the external environment. Frequently their receptors overlap both in the intracellular signaling pathways that they use and the effects that they mediate (). For example, receptors for both IL-6 and for the adipose tissue-derived hormone leptin signal through the Jak2-Stat3 signaling pathway () and both molecules promote proliferation of immune cells and excretion of cytokines by macrophages (). The insulin receptor shares its downstream signaling cascade with CD28, one of the most potent costimulatory molecules for CD8T cells (). Both pathways converge on phosphatidylinositol 3 kinase (PI3K), enhancing anabolic metabolism and increasing glucose transporter amounts on the cell membrane (). IR is associated with hyperinsulinemia. Whether insulin plays a role in promoting immune responses following infection is unknown.

Diabetes mellitus type 2 (DM2) is a highly prevalent () metabolic disease, characterized by high blood glucose concentrations. The pathology of DM2 involves many organs but its main underlying mechanism is decreased insulin sensitivity of the liver and skeletal muscle and an inability of pancreatic β-cells to compensate for this defect (). DM2 is diagnosed based on increased concentrations of glycosylated hemoglobin (HbA1c), fasting plasma glucose (FPG) or 2-hr postprandial blood glucose (2HG) (). If these concentrations are increased, but do not reach DM2 threshold values, people are diagnosed with pre-diabetes (). Prospective studies show that changes in glycemic control occur gradually over years, but typically contain an abrupt increase in metabolic parameters preceding diagnosis of DM2 (). Progression from pre-diabetes to DM2 therefore appears to involve an unknown “event” that pushes systemic insulin resistance (IR) beyond the ability of the pancreas to compensate. DM2 is associated with chronic systemic low-grade inflammation originating in visceral adipose tissue (VAT) (). Obese VAT accumulates pro-inflammatory immune cells and drives a type-1 immune response, normally associated with viral infection, characterized by the production of cytokines such as tumor necrosis factor (TNF) and interleukin-1β (IL-1β) (). Obesity thus mimics a state of chronic systemic low-grade infection and leakage of pro-inflammatory cytokines into circulation is thought to contribute to systemic IR (). Acute infection might therefore represent the “event” that drives rapid progression to DM2 in pre-diabetes. Only few epidemiological studies address this topic and these do suggest that infection is associated with a higher risk of DM2 (). However, direct experimental evidence if and how infection impacts glycemic control are lacking.

Cytomegalovirus seropositivity is associated with glucose regulation in the oldest old. Results from the Leiden 85-plus Study.

Banting Lecture. From the triumvirate to the ominous octet: a new paradigm for the treatment of type 2 diabetes mellitus.

To test the importance of insulin on CD8T cell priming in a second model, we generated Ins2iDTR mice, which allow elimination of insulin-producing pancreatic beta cells by injection of diphtheria toxin (DT). DT-treatment of Ins2iDTR abrogated their ability to produce insulin in response to a glucose bolus ( Figures S7 A–S7C), but did not result in overt morbidity at least one week after treatment. DT-treated Ins2iDTR or iDTR littermates were infected with MCMV and CD8T cell responses were analyzed one week later. DT-treatment caused reduction in the number of virus-specific CD8T cells upon MCMV infection and impaired their cytokine production ( Figures 7 C and 7D). Finally, after MCMV infection, DT-treated Ins2iDTR mice had reduced capacity to kill viral peptide-pulsed target cells ( Figure 7 E). To confirm that virus-induced muscle IR was responsible for the enhanced CD8T cell response we infected Ifngr1mice and littermate controls with MCMV and analyzed CD8T cell responses 1 week later. Prevention of skeletal muscle IR caused reduction in the number KLRG1virus-specific CD8T cells ( Figure 7 F). Moreover, CD8T cells showed impaired cytokine production upon in vitro re-stimulation ( Figure 7 G). Thus, we have identified insulin as a molecule which promotes antiviral-effector CD8T cell responses.

To confirm these findings in vivo in an obesity-independent model of hyperinsulinemia, lean mice were injected daily with basal (long-acting) insulin. Unlike short-working insulin used in ITT, basal insulin is slowly released in the blood stream and causes a continuous state of hyperinsulinemia. Basal insulin-treated animals were infected with MCMV and CD8T cell responses were analyzed after 7 days. We observed that hyperinsulinemia promoted effector cell formation and cytokine production of virus-specific CD8T cells ( Figures 7 A and 7B ).

(F and G) Ifngr1mice and Ifngr1littermates were infected with MCMV. After 7 days (F) effector cells (KLRG1) specific for two MCMV epitopes were quantified in spleen. In addition, (G) splenocytes were re-stimulated in vitro with two different viral peptides and production of IFN-γ and TNF was analyzed by flow cytometry. For (A–E) five mice and for (F and G) eight mice were used in each group. Representative of two or more experiments is shown. Indicated are means ± SEM and statistical significances atp < 0.05,p < 0.01,p < 0,001 by (A–G) Student’s t test. p.i., post infection. See also Figure S7

(C–E) Ins2 cre Rosa iDTR and Rosa iDTR littermates were DT-treated and subsequently infected with MCMV. After 7 days (C) absolute numbers of antigen-specific cells were determined in spleen. (D) Splenocytes were re-stimulated in vitro with viral peptides and cytokine production was analyzed by flow cytometry. (E) Mice were injected with splenocytes pulsed with control, M57, or m139 peptides, labeled with different CFSE concentrations. After 4 hr, specific killing was determined in spleen.

(A and B) B6 mice were infected with MCMV and treated daily with basal insulin or PBS. Seven days p.i. (A) effector cells (KLRG1 + ) specific for two MCMV epitopes were quantified in spleen. Histograms show representative plots. Gated is for CD8 + tetramer + cells. (B) Splenocytes were re-stimulated in vitro with indicated peptides and TNF production was analyzed by flow cytometry.

Finally, we questioned the physiological relevance of IFN-γ-induced IR. In lean animals, infection did not induce hyperglycemia, excluding increased nutrient availability as a cause. However, viral infection did cause hyperinsulinemia. Because insulin receptor and CD28 signaling both converge on PI3K (), we hypothesized that insulin might directly provide co-stimulation for CD8T cells. We observed that CD8T cells expressed both Insr and Irs2, but not Irs1 ( Figure 6 A). Indeed, stimulation of primed CD8T cells with insulin rapidly induced phosphorylation of S6 kinase ( Figure 6 B), a downstream target of insulin-signaling. Next, we stimulated OT-1 CD8T cells in vitro with SIINFEKL peptides and/or αCD28 antibodies in the presence or absence of insulin. Proliferation and viability were not affected by insulin ( Figures S6 A and S6B). In contrast, cytokine and Granzyme B production were enhanced by insulin, especially upon CD28 co-stimulation ( Figure 6 C).

(C) Purified OT-1 cells were stimulated with N4 peptide alone or in the presence of insulin and/or anti-CD28. After 48 hr, cells were re-stimulated with N4 peptide and production of Granzyme B, IFN-γ, and TNF was measured by flow cytometry. For (A–C) a representative of at least three experiments is shown. For (A)–(C) at least three samples were used in each group Indicated are means ± SEM and statistical significances atp < 0.05,p < 0.01,p < 0,001 by (C) ANOVA followed by Bonferroni post-testing. See also Figure S6

(B) Purified OT-1 cells were stimulated with SIINFEKL (N4) peptide in the presence or absence of anti-CD28. After 2 days, cells were rested from stimuli for 3 hr, followed by stimulation with 1 U/ml of insulin. Kinetics of S6 phosphorylation were determined by flow cytometry. Representative plot shows cells primed with N4 and αCD28 at 0 and 15 min after insulin stimulation.

(A) qPCR was used to quantify expression of Insr, Irs2, and Irs1 in purified OT-1 CD8 + T cells. Expression was normalized to Hprt.

Taken together, these results show that infection-induced IFN-γ drives IR in skeletal muscles through downregulation of the insulin receptor, resulting in GI in pre-diabetic animals.

Because MCMV infection has a long-lasting negative effect on insulin sensitivity, at least in pre-diabetic mice, we wanted to elucidate whether 3 weeks p.i. Insr is still downregulated in muscle. We observed that transcription of Insr in muscle of HFD primed MCMV infected mice are still downregulated at this time point ( Figure 5 F).

Next, we sought to elucidate how infection-induced IFN-γ drives IR in muscle. Infection did not affect total Akt protein expression, suggesting that IFN-γ impairs upstream insulin receptor signaling. No differences were observed in transcription of Irs1 and Irs2 ( Figure S5 A). Also, we found no increase in Socs1 or Socs3, which are known targets of IFN-γ and known inhibitors of insulin signaling () ( Figure S5 B). In contrast, transcription of Insr, coding for the insulin receptor, was significantly reduced in muscle, but not in liver of infected pre-diabetic mice ( Figure 5 A). Moreover, we found that infection of WT, but not Ifngmice with MCMV resulted in downregulation of insulin receptor expression on transcriptional and protein amounts in muscle ( Figure 5 B–5D; S5C and S5D ). Likewise, infection with LCMV or Influenza A also resulted in downregulation of Insr in muscle ( Figure S5 E). To investigate whether IFN-γ alone induces downregulation of Insr or whether this effect is only achieved in the context of viral infection, we injected IFN-γ daily in the m. sartorius of NCD or HFD primed mice. We observed that IFN-γ injection alone was able to cause upregulation of MHC II, a bona fide downstream target of IFNγR signaling, on muscle tissue macrophages ( Figure S5 F) and downregulation of Insr transcript in muscle but not in the liver ( Figure 5 E, S5 G).

(F) Prediabetic or NCD fed mice were infected with MCMV. 3 weeks p.i. Insr expression in muscle was determined by qPCR. Representative of two (E and F) or three (A–D) experiments is shown. For (A)–(F), at least four mice were used in each group. Indicated are means ± SEM and statistical significances atp < 0.05,p < 0.01,p < 0,001 by (A, E, F) ANOVA followed by Bonferroni post-testing or (B–D) Student’s t test. p.i., post infection. See also Figure S5 . InsR, protein expression.

(E) Pre-diabetic or NCD fed mice were injected daily in one m. sartorius with mouse recombinant IFN-γ or PBS. After 3 days, transcript of Insr were determined in m. sartorius. R (right), site of injection; L (left), collateral symmetric muscle.

(C and D) Ifng −/− mice were MCMV or mock infected and after 5 days skeletal muscle samples were isolated and (C) Insr expression was determined by qPCR. Expression was normalized to Hprt.

(B) Skeletal muscle samples were isolated 7 days after MCMV infection of pre-diabetic mice, and protein expression of insulin receptor was determined by immunoblot.

(A) Pre-diabetic or NCD fed mice were infected with MCMV or left untreated. On day 7 after infection, Insr transcripts were determined in muscle and liver by qPCR. Expression was normalized to Hprt.

The comparative roles of suppressor of cytokine signaling-1 and -3 in the inhibition and desensitization of cytokine signaling.

These results demonstrate that skeletal muscle cells are the main targets of IFN-γ induced IR and progression of DM2 following infection.

Next, we considered skeletal muscle as a target tissue. Skeletal muscle is responsible for 70%–75% of insulin-induced glucose absorption (). We noticed that infection, but not DIO increases IFN-γ transcription in skeletal muscle 7 days post infection ( Figure S4 ). Therefore, we hypothesized that infection directly targets insulin sensitivity of muscle cells. Indeed, we observed that infection reduced pAKT in muscle of lean and obese animals upon insulin challenge ( Figure 4 A). This effect was IFN-γ-dependent, since we did not observe this effect in Ifngmice ( Figure 4 B). Moreover, CkmmIfngr1mice (Ifngr1), which lack the IFNγR1 receptor on myocytes, were protected from infection-induced IR and GI ( Figure 4 C). To demonstrate that infection specifically impairs glucose uptake into skeletal muscle in response to insulin, mice were subjected to hyperinsulinemic-euglycemic clamping and were injected with a bolus of radioactive 2-deoxy glucose at the end of the steady state period. We noted that glucose uptake in muscle was strongly reduced following infection, whereas internalization in VAT was not affected ( Figure 4 D).

(D) Glucose uptake into VAT and skeletal muscle during hyperinsulinemic-euglycemic clamping 5 days after MCMV infection of NCD fed mice. The experiment in (D) was performed once. In (A) and (B) density quantification plot shows pooled data from two independent experiments. For (C) a representative of three experiments is shown. For (A)–(D), five mice per experiment were used in each group. Indicated are means ± SEM and statistical significances atp < 0.05,p < 0.01,p < 0,001 by (D) Student’s t test or (A–C) ANOVA followed by Bonferroni post-testing. p.i., post infection. See also Figure S4

(C) Pre-diabetic Ifngr1 ΔMyo mice and Ifngr1 flox/flox littermates were injected with MCMV or PBS. 5 and 7 days p.i., mice were subjected to ITT and GTT.

(A and B) B6 and Ifng −/− mice were NCD or HFD-fed for 6 weeks, followed by MCMV infection. After 7 days mice were fasted overnight, followed by injection with insulin. After 30 minutes, skeletal muscle samples were isolated and pan-Akt and pAkt amounts were determined by immunoblotting.

To investigate whether infection reduces insulin sensitivity of the liver, we infected pre-diabetic mice with MCMV. One-week p.i. we observed that DIO, but not infection increased FPG concentrations ( Figure 3 H). In addition, one-week p.i. we analyzed gluconeogenesis by pyruvate tolerance test (PTT). In line with FPG concentrations, we observed that HFD increased gluconeogenesis following pyruvate challenge. However, infection did not result in enhanced, but even in somewhat reduced gluconeogenesis ( Figure 3 I). In addition, hepatocytes from HFD-fed mice showed reduced induction of AKT phosphorylation (pAKT) in response to insulin challenge compared to NCD-fed mice, whereas infection did not further impair this process ( Figure 3 J). Finally, hepatocyte-specific ablation of the IFNγR1 using AlbIfngr1animals (Ifngr1) did not result in a reduction of IR or GI following infection of pre-diabetic mice ( Figure S3 C). To confirm that infection does not result in hepatic insulin resistance, we calculated the endogenous glucose production rate (EGP) under basal conditions and after infusion of insulin during a hyperinsulinemic-euglycemic clamp study. We did not observe an impact of infection on EGP under either condition ( Figure 3 K). Thus, infection-induced IR and GI is mediated independently of macrophages, adipocytes, and hepatocytes.

We therefore considered that IFN-γ directly affects insulin sensitivity of one or more organs involved in glucose homeostasis. In the DIO model, VAT is the main source of chronic systemic inflammation and plays a key role in the development of IR and GI. We have shown previously that surgical removal of VAT (VATectomy) prevents development of GI and IR in non-infected obese mice (). We performed VATectomy 2 weeks before initiation of HFD feeding and MCMV infection. GTT was performed 10 weeks after the surgery. As expected, VATectomy reduced glucose intolerance in non-infected animals. In contrast, removal of visceral fat pads was not able to prevent infection-induced GI ( Figure 3 E). Since VATectomy does not remove all adipocyte deposits in mice, we conditionally ablated IFNγR1 from adipocytes. Pre-diabetic AdipoqIfngr1mice (Ifngr1) were not able to prevent infection-induced IR or GI in comparison to littermate controls ( Figures 3 F and 3G). Thus, adipocytes do not play a major role in infection-induced GI in pre-diabetic mice.

Previously we showed that NK-cell-derived IFN-γ promotes IR in the DIO model by inducing M1 adipose tissue macrophage (ATM) polarization (). We hypothesized that this mechanism also operates in the context of infection. Whereas infection does enhance ATM conversion and tissue inflammation, clodronate-mediated neutralization of these cells did not prevent infection-induced GI ( Figures S3 A and S3B). To confirm that IFN-γ mediates its effect in infected mice independently of macrophages, we conditionally ablated the receptor for IFN-γ (IFN-γR1) on these cells. Lyz2Ifngr1(Ifngr1) and littermate controls were placed on HFD for 6 weeks and then infected with MCMV. Five days p.i. we observed no difference in insulin sensitivity ( Figure 3 C) or GI ( Figure 3 D) between Ifngr1and littermate controls. Thus, IFN-γ mediates its effect on IR and GI in infected pre-diabetic mice independently of macrophages.

To investigate whether systemic or local increase of IFN-γ concentrations is responsible for the effect, we infected pre-diabetic mice with MCMV and plasma IFN-γ concentrations were followed over time. We observed that increased blood IFN-γ returned to baseline at time points when it still impacted GI and IR ( Figure 3 A). This result suggests that IFN-γ either has a long-lasting effect on the ability of tissues to sense insulin or that there is a local source of IFN-γ that sustains impairment of insulin sensitivity. We neutralized IFN-γ starting 7 days post infection (p.i.) of HFD-primed mice, when its concentrations in the blood have returned to baseline. GI was completely prevented in treated mice ( Figure 3 B), which indicates that a local source of IFN-γ drove continued GI and IR in pre-diabetic mice following infection.

(K) B6 mice were mock-infected or infected with MCMV and endogenous glucose production was analyzed 5 days later during hyper-insulinemic-euglycemic clamp. (H) Pooled data of 12 experiments (n = 60) is shown (analyzed by ANOVA with Bonferroni post-testing). The experiment in Figure 3 K was performed once. Other graphs show one of two or more experiments with similar results. For (A–G) and (I–K) five mice were used in each group. Indicated are means ± SEM. and statistical significances atp < 0.05,p < 0.01,p < 0,001 by student`s t test or ANOVA followed by Bonferroni post-testing. p.i., post infection. See also Figure S3

(H–J) Mice were fed for 6 weeks with indicated diets, followed by MCMV or PBS injection. (H) FPG was determined 7 days after infection. (I) PTT was performed on day 7 after infection. (J) 7 days p.i., mice were fasted overnight, followed by injection with insulin. After 30 minutes, liver samples were isolated and pan-Akt and pAkt amounts were determined by Immuno blotting.

(F and G) Pre-diabetic Ifngr1 ΔAdi mice and Ifngr1 flox/flox littermates were injected with MCMV or PBS. 5 and 7 days after infection mice were subjected to (F) ITT or (G) GTT, respectively.

(E) Mice either underwent sham operation or surgical excision of periepididymal fat pads (VATectomy). 2 weeks after surgery, mice were infected with MCMV and placed on HFD. GTT was performed after 8 weeks.

(C and D) Pre-diabetic Ifngr1 ΔMac mice and Ifngr1 flox/flox littermates were injected with MCMV or PBS. 7 days p.i. mice were subjected to (C) ITT or (D) GTT.

(B) Pre-diabetic mice were MCMV or mock infected and treated with neutralizing mAbs to IFN-γ every 3 days starting 1 day before or 7 days p.i. Fourteen days p.i. mice were subjected to GTT.

(A) Serum IFN-γ concentrations were determined by ELISA at different time points after infection of mice primed with HFD or NCD for 6 weeks.

In summary, virally-induced IFN-γ promotes IR and GI and drives the rapid progression from pre-diabetes to DM2 in infected mice.

IFN-γ is produced exclusively by immune cells () following viral infection, in particular by NK cells, CD4and CD8T cells. We depleted NK, CD4, or CD8T cells in MCMV infected, pre-diabetic mice ( Figures 2 E and 2F). Only elimination of NK cells resulted in a loss of GI ( Figure 2 F). To investigate whether NK cells have a prolonged effect on glycemic control in obese animals, we infected NK cell-depleted mice simultaneously with the start of HFD. Depletion of NK cells completely prevented development of GI 8 weeks after infection ( Figure S2 M). To confirm that NK cells drive GI through IFN-γ, Ifngmice received PBS or WT NK cells preceding MCMV infection. Only in the presence of WT NK cells, did Ifngmice develop IR ( Figure 2 G). In addition, infection of HFD primed mice with m157-deficient MCMV virus (Δm157), which precludes Ly49h-mediated activation of NK cells, did not induce development of GI ( Figure S2 N). Thus, MCMV infection drives development of GI and IR in pre-diabetic mice via NK cell-derived IFN-γ.

We therefore considered the possibility that the immune system drives IR through specific cytokines following infection. Viral infection activates the inflammasome (), which generates the pro-diabetic cytokine IL-1β (). However, neither chemical inhibition of the NLRP3 inflammasome activity, nor neutralization of IL-1β with antibodies resulted in amelioration of GI following MCMV infection of pre-diabetic mice ( Figures S2 E and S2F). In fact, neutralization of IL-1β increased glucose intolerance following infection, which corresponds with the previously reported positive effect of this cytokine on glycemic control (). Type-1 inflammatory cytokines such as interferons and TNF have a negative impact on glucose homeostasis in the context of obesity (). We therefore injected lean and pre-diabetic mice with poly I:C, a strong inducer of type-1 interferons (). Whereas this treatment did activate peritoneal macrophages, no impact on GI was observed ( Figure S2 G and S2H). TNF has been shown to induce IR in ob/ob mice through TNF receptor 1 (TNFR1) (). However, deficiency for TNFR1 did not result in amelioration of GI following MCMV infection of pre-diabetic animals ( Figure S2 I). In contrast, although neutralization of interferon-γ (IFN-γ) was associated with increased viral titers in some tissues important for glucose homeostasis ( Figure S2 J), it completely prevented GI and IR in MCMV and LCMV infected pre-diabetic animals ( Figures 2 C and 2D; S2 K). Similar results were achieved in Ifngmice ( Figure S2 L). This data shows that IFN-γ plays a dominant role in progression of DM2 induced by different viruses.

The liver has an important role in maintaining euglycemia through gluconeogenesis, glycogenolysis, and glycogenesis. Liver diseases, including viral hepatitis, are therefore frequently associated with aberrations in glucose homeostasis (). To investigate whether liver damage alone may be responsible for enhanced progression of GI in our model, we exposed pre-diabetic mice to the hepatotoxic compounds CCL) or paracetamol () ( Figures S2 A and S2B). Neither compound induced development of GI in pre-diabetic animals ( Figure S2 C and S2D).

New data and new concepts on the role of the liver in glucose homeostasis.

[Role of the liver in the regulation of glucose metabolism in diabetes and chronic liver disease].

To gain insight in the mechanism underlying virus-induced progression of DM2 we first investigated whether viral infection affects glucose sensing by the pancreas after HFD priming. We observed that infection of pre-diabetic mice with MCMV resulted in increased FPI and enhanced insulin production following glucose challenge ( Figure 2 A). To further prove that MCMV infection increases insulin secretion we calculated the insulinogenic index, which positively correlates with insulin output from pancreatic β-cells (). We observed that the insulinogenic index was increased in infected, HFD-fed animals compared to controls, excluding pancreatic dysfunction as a cause for GI ( Figure 2 B).

(A–F) Mice were fed for 6 weeks with indicated diets, followed by MCMV infection. Control mice were treated with PBS. (A) 7 days p.i., mice were fasted overnight and challenged with glucose. Insulin concentrations were determined in serum by ELISA. (B) Insulinogenic index during GTT. (C and D) Infected pre-diabetic or NCD-fed mice were injected every 3 days with neutralizing mAbs to IFN-γ or with isotype matched irrelevant mAbs starting 24 hr before infection. (C) GTT and (D) ITT were performed on day 7 after infection. Infected pre-diabetic mice were injected with (E) CD4- or CD8-depleting or (F) NK-cell-depleting antibodies. Control animals were treated with isotype matched irrelevant mAbs. GTT was performed on day 7 p.i. (G) B6 or Ifngmice were infected with MCMV. Ifngmice were transferred with NK cells or PBS 1 day before infection and 3 days later. 5 days p.i. mice were subjected to ITT. For (A)–(G) a representative of three experiments is shown. For (A–G) five mice were used in each group. Indicated are means ± SEM. and statistical significances atp < 0.05,p < 0.01,p < 0,001 by ANOVA followed by Bonferroni post-testing. p.i., post infection. See also Figure S2

In summary, viral infection of “pre-diabetic” obese mice causes a reduction of glycemic control and aggravates development of clinical complications associated with DM2.

Our findings indicate that viral infection is an independent risk factor for development of diabetes by pre-diabetic obese individuals. To investigate whether we could therapeutically prevent GI in pre-diabetic mice, animals were treated with the antiviral drug ganciclovir starting one day after infection. Ganciclovir treatment strongly reduced viral replication ( Figure S1 O) and prevented development of virus-induced glucose intolerance in HFD primed mice ( Figure 1 Q).

Chronic, uncontrolled DM2 is associated with development of microvascular and macrovascular complications, such as diabetic nephropathy (DN). To investigate whether infection aggravates development of DM2-associated complications, animals were infected with MCMV on the same day when we started with HFD feeding. At 16 weeks, infection of pre-diabetic mice promoted hypertrophy of juxtamedullary glomeruli, an early sign of DN () ( Figure 1 N). After 24 weeks, histological analysis revealed more severe symptoms of DN in 10%–20% of glomeruli of infected HFD-fed mice, but not in lean or uninfected animals, whereas kidneys from obese mice were mildly affected in only 5% of glomeruli ( Figure 1 O; S1 M). 24 weeks after infection, viral replication was not detectable in kidneys of mice fed either with NCD or HFD, indicating that virus-induced pathology is not directly responsible for DN ( Figure S1 N). In addition, infection with MCMV of HFD fed animals resulted in increased thickness of the basement membrane, another bona fide marker of DN ( Figure 1 P).

An important question from the perspective of clinical relevance is whether the impact of infection on glycemic control is transient or permanent. Indeed, we observed that three weeks after MCMV infection, pre-diabetic mice still showed increased GI ( Figure 1 K) although there was no difference in viral titers between normal chow diet (NCD) or HFD fed animals ( Figure S1 J), suggesting a long-lasting effect. When MCMV infection and HFD feeding were started on the same day, increased IR and reduced glycemic control were observed for at least 8 weeks in infected animals, compared to non-infected controls ( Figures 1 L and 1M). In contrast, the impact of Influenza A or LCMV infection on glucose intolerance of HFD-primed animals appeared to be transient ( Figure S1 K and S1L).

Systemic IR is the underlying cause of DM2. However, people are only diagnosed as diabetics when systemic IR has reached a level at which compensatory mechanisms fail to lower blood glucose concentrations below well-defined thresholds. Thus, DM2 is diagnosed by measuring GI, rather than IR. In pre-diabetes, frequently an unknown “event” drives rapid development of DM2 (). To see whether infection represents such an event, animals were placed on a diet with high fat-content (HFD), resulting in diet-induced obesity (DIO). HFD generates systemic IR and GI in 3 months (), but after 6 weeks results only in hepatic IR, characterized by increased FPG and pyruvate intolerance ( Figure S1 C and S1D), but not yet systemic IR or GI ( Figures S1 E and S1F), thus resembling human pre-diabetes (). Infection of 6 weeks HFD-primed (“pre-diabetic”) mice with MCMV resulted in both IR and GI ( Figures 1 I and 1J), but did not affect obesity ( Figure S1 G). To test whether other viral pathogens also affect glucose homeostasis, we infected pre-diabetic mice with lymphocytic choriomeningitis (LCMV) or influenza A virus. Both infections resulted in similar loss of glucose control as observed after MCMV infection ( Figures S1 H and S1I).

Taken together, human and mouse data show that infection transiently induces IR, but does not result in overt GI due to compensatory hyperinsulinemia.

We next used murine cytomegalovirus (MCMV) as an animal model for a common human infection (). MCMV has a broad tropism, including key organs involved in the regulation of glucose homeostasis, such as liver, VAT, pancreas, and skeletal muscle ( Figure 1 D). Animals were infected with MCMV and after 7 days subjected to an insulin tolerance test (ITT). We observed that MCMV infection resulted in transient intolerance to insulin ( Figure 1 E; S1 A). To confirm that this effect was the result of IR, mice were subjected to hyperinsulinemic-euglycemic clamping on day 5 after infection. Indeed, infected animals showed a strongly reduced sensitivity to insulin in comparison to non-infected controls ( Figure 1 F; S1 B), as determined by a lower glucose infusion rate (GIR). To determine whether infection-induced IR resulted in loss of glycemic control, we analyzed infected animals by glucose tolerance test (GTT). Despite IR, infection did not result in GI ( Figure 1 G). As in humans, infection was associated with elevated FPI concentrations. Moreover, following glucose challenge, increased insulin production was observed in infected animals, explaining why systemic IR does not result in GI ( Figure 1 H).

First, we investigated whether infection impacts systemic metabolic parameters in humans. Body mass index (BMI) and blood parameters were obtained from euglycemic people with normal weight (BMI of 18–25 kg/m) and overweight (BMI > 25), diagnosed with acute respiratory infection at time of presentation of symptoms and three months later. Infection transiently increased fasting plasma insulin (FPI) in both groups ( Figure 1 A). In contrast, FPG was not significantly affected by infection ( Figure 1 B). Notably, the homeostasis model assessment—insulin resistance (HOMA-IR) index values, which inversely correlate with systemic insulin sensitivity, were increased during acute infection, especially for people with overweight ( Figure 1 C). Thus, acute infection appeared to transiently decrease systemic insulin sensitivity in humans, without affecting blood glucose concentrations.

(Q) MCMV infected pre-diabetic mice were treated daily with ganciclovir starting 24 h p.i. or with PBS. After 7 days, mice were subjected to GTT. The experiment in (F) was performed once. For all other experiments, a representative of three experiments is shown. For (D)–(Q), five mice were used in each group. Indicated are means ± SEM and statistical significances atp < 0.05,p < 0.01,p < 0,001 by (A–C, E–H) Student’s t test or (I–Q) ANOVA followed by Bonferroni post-testing. p.i., post infection. See also Figure S1

(L–P) Mice were infected with MCMV simultaneously with the start of HFD. 8 weeks p.i. mice were subjected to (L) GTT and (M) ITT. In addition, (N), sizes of juxtamedullary glomeruli were determined 16 weeks p.i. (n = 5; 72 glomeruli per animal). 24 weeks p.i. (O) PAS staining of kidney sections was performed. (Green arrow) expansion of mesangial matrix and (yellow arrow) increased Bowman capsule size or (P) thickness of basement membrane are shown.

(I–K) Mice were placed on NCD or HFD for 6 weeks (“pre-diabetic” mice) before infection with MCMV or PBS. 1 week later, they were subjected to (I) ITT or (J) GTT. (K) GTT was also performed 3 weeks after infection.

(G and H) B6 mice were mock-infected or infected with MCMV and analyzed 7 days later by (G) GTT and (H) serum insulin concentrations after glucose challenge upon overnight fasting were measured. In addition, area under curve is shown.

(E) B6 mice were mock-infected or infected with MCMV and analyzed 7 days later by ITT. (F) B6 mice were mock-infected or infected with MCMV and analyzed 5 days later by hyperinsulinemic-euglycemic clamp. Glucose infusion rate (GIR) is shown. n = 5.

(D) MCMV titters (PFU) in indicated tissues of B6 mice at day 4, 7, and 10 p.i. are shown. LD, limit of detection.

Patients with respiratory infection were segregated in two groups (normal weight: BMI < 25 [n = 17], overweight: BMI > 25 [n = 14]) and analyzed at time of diagnosis and after 3 months for (A) FPI, (B) FPG, and (C) HOMA-IR index.

Discussion

Our research has addressed the question how viral infection contributes to development of DM2. We found that the activated immune system drove systemic IR in response to infection with various viruses, but not GI due to compensatory insulin output by the pancreas. In case of pre-existing metabolic dysfunction caused by DIO, compensatory mechanisms were overloaded and long-term loss of glycemic control ensued. We discovered that virally-induced IFN-γ directly and specifically targeted skeletal muscle to downregulate the insulin receptor and promoted compensatory hyperinsulinemia to boost the CD8+ T cell-mediated antiviral immune response. Thus, here we have identified a physiological feed-back mechanism between the immune and endocrine systems, which operates in viral infection and we demonstrate that this mechanism represents an “Achilles heel” for deregulation of glycemic control in pre-diabetic obese subjects.

+ T cells through the activation of several signaling cascades, including the PI3K pathway ( Rudd et al., 2009 Rudd C.E.

Taylor A.

Schneider H. CD28 and CTLA-4 coreceptor expression and signal transduction. + T cells during priming enhanced the costimulatory effects of CD28 and functioned as a pro-inflammatory cytokine. Immune cell-mediated increase of insulin availability at the time of CD8+ T cells priming, therefore benefits the immune response. However, immune-mediated adjustment of insulin production comes at a risk of inducing loss of glucose homeostasis and should therefore be carefully regulated. For example, if IFN-γ would directly stimulate pancreatic insulin production, it would induce potentially lethal hypoglycemia. Similarly, induction of IR in liver or VAT would result in an increase of FPG or circulating free fatty acids, respectively ( Karpe et al., 2011 Karpe F.

Dickmann J.R.

Frayn K.N. Fatty acids, obesity, and insulin resistance: time for a reevaluation. Leto and Saltiel, 2012 Leto D.

Saltiel A.R. Regulation of glucose transport by insulin: traffic control of GLUT4. Meshkani and Adeli, 2009 Meshkani R.

Adeli K. Hepatic insulin resistance, metabolic syndrome and cardiovascular disease. Wang et al., 2016 Wang A.

Huen S.C.

Luan H.H.

Yu S.

Zhang C.

Gallezot J.D.

Booth C.J.

Medzhitov R. Opposing Effects of Fasting Metabolism on Tissue Tolerance in Bacterial and Viral Inflammation. Dror et al., 2017 Dror E.

Dalmas E.

Meier D.T.

Wueest S.

Thévenet J.

Thienel C.

Timper K.

Nordmann T.M.

Traub S.

Schulze F.

et al. Postprandial macrophage-derived IL-1β stimulates insulin, and both synergistically promote glucose disposal and inflammation. CD28 provides one of the strongest co-stimulatory signals for activation of CD8T cells through the activation of several signaling cascades, including the PI3K pathway (). The insulin receptor exclusively signals through PI3K, thus sharing a major signaling cascade with CD28. Indeed, we found that insulin stimulation of CD8T cells during priming enhanced the costimulatory effects of CD28 and functioned as a pro-inflammatory cytokine. Immune cell-mediated increase of insulin availability at the time of CD8T cells priming, therefore benefits the immune response. However, immune-mediated adjustment of insulin production comes at a risk of inducing loss of glucose homeostasis and should therefore be carefully regulated. For example, if IFN-γ would directly stimulate pancreatic insulin production, it would induce potentially lethal hypoglycemia. Similarly, induction of IR in liver or VAT would result in an increase of FPG or circulating free fatty acids, respectively (). By targeting insulin sensitivity of muscle, increased insulin production by the pancreas both boosts the antiviral immune response, while ensuring blood glucose homeostasis. Muscle IR could cause reduced motility, but this is in fact a desirable behavior upon infection (). Recently, it has been shown that under homeostatic conditions macrophage-derived IL-1β can promote insulin secretion which stimulates glucose uptake in immune cells (). Our findings indicate that IL-1β does not reduce systemic glucose uptake following infection. However, neutralization of IL-1β resulted in enhanced glucose intolerance following infection, suggesting that immune-endocrine interactions operate at multiple levels to increase systemic insulin concentrations.

+ T cells and M1 macrophages. Cytokines such as TNF, IL-6, and IL-1β produced by these cells leak into circulation and induce insulin resistance ( Johnson et al., 2012 Johnson A.R.

Milner J.J.

Makowski L. The inflammation highway: metabolism accelerates inflammatory traffic in obesity. O’Rourke et al., 2012 O’Rourke R.W.

White A.E.

Metcalf M.D.

Winters B.R.

Diggs B.S.

Zhu X.

Marks D.L. Systemic inflammation and insulin sensitivity in obese IFN-γ knockout mice. Wensveen et al., 2015a Wensveen F.M.

Jelenčić V.

Valentić S.

Šestan M.

Wensveen T.T.

Theurich S.

Glasner A.

Mendrila D.

Štimac D.

Wunderlich F.T.

et al. NK cells link obesity-induced adipose stress to inflammation and insulin resistance. Grzelkowska-Kowalczyk and Wieteska-Skrzeczyńska, 2009 Grzelkowska-Kowalczyk K.

Wieteska-Skrzeczyńska W. Treatment with IFN-gamma prevents insulin-dependent PKB, p70S6k phosphorylation and protein synthesis in mouse C2C12 myogenic cells. McGillicuddy et al., 2009 McGillicuddy F.C.

Chiquoine E.H.

Hinkle C.C.

Kim R.J.

Shah R.

Roche H.M.

Smyth E.M.

Reilly M.P. Interferon gamma attenuates insulin signaling, lipid storage, and differentiation in human adipocytes via activation of the JAK/STAT pathway. Wada et al., 2011 Wada T.

Hoshino M.

Kimura Y.

Ojima M.

Nakano T.

Koya D.

Tsuneki H.

Sasaoka T. Both type I and II IFN induce insulin resistance by inducing different isoforms of SOCS expression in 3T3-L1 adipocytes. The role of obesity-induced visceral adipose tissue inflammation in development of IR has been well described. Adipocyte hypertrophy promotes accumulation of pro-inflammatory cells with a Th1 cell-signature, such as CD8T cells and M1 macrophages. Cytokines such as TNF, IL-6, and IL-1β produced by these cells leak into circulation and induce insulin resistance (). We and others have shown that NK-cell-derived IFN-γ promotes development of IR indirectly by driving conversion of adipose tissue macrophages (ATMs) toward an M1 phenotype (). Infection-induced IR appears to operate independently of VAT. Moreover, we have demonstrated in vivo that IFN-γ is able to induce IR directly in non-immune cells. In vitro, IFN-γ had been shown to cause IR in 3T3-L1 adipocytes and myoblasts by inhibiting insulin receptor signaling through induction of suppressor of cytokine signaling (SOCS) molecules (). We show that in vivo IFN-γ specifically reduced glucose uptake in skeletal muscle by downregulation of the insulin receptor.