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, 7 (10), e46824

Non-diabetic Hyperglycemia Exacerbates Disease Severity in Mycobacterium Tuberculosis Infected Guinea Pigs

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Non-diabetic Hyperglycemia Exacerbates Disease Severity in Mycobacterium Tuberculosis Infected Guinea Pigs

Brendan K Podell et al. PLoS One.

Abstract

Hyperglycemia, the diagnostic feature of diabetes also occurs in non-diabetics associated with chronic inflammation and systemic insulin resistance. Since the increased risk of active TB in diabetics has been linked to the severity and duration of hyperglycemia, we investigated what effect diet-induced hyperglycemia had on the severity of Mycobacterium tuberculosis (Mtb) infection in non-diabetic guinea pigs. Post-prandial hyperglycemia was induced in guinea pigs on normal chow by feeding a 40% sucrose solution daily or water as a carrier control. Sucrose feeding was initiated on the day of aerosol exposure to the H37Rv strain of Mtb and continued for 30 or 60 days of infection. Despite more severe hyperglycemia in sucrose-fed animals on day 30, there was no significant difference in lung bacterial or lesion burden until day 60. However the higher spleen and lymph node bacterial and lesion burden at day 30 indicated earlier and more severe extrapulmonary TB in sucrose-fed animals. In both sucrose- and water-fed animals, serum free fatty acids, important mediators of insulin resistance, were increased by day 30 and remained elevated until day 60 of infection. Hyperglycemia mediated by Mtb infection resulted in accumulation of advanced glycation end products (AGEs) in lung granulomas, which was exacerbated by sucrose feeding. However, tissue and serum AGEs were elevated in both sucrose and water-fed guinea pigs by day 60. These data indicate that Mtb infection alone induces insulin resistance and chronic hyperglycemia, which is exacerbated by sucrose feeding. Moreover, Mtb infection alone resulted in the accumulation tissue and serum AGEs, which are also central to the pathogenesis of diabetes and diabetic complications. The exacerbation of insulin resistance and hyperglycemia by Mtb infection alone may explain why TB is more severe in diabetics with poorly controlled hyperglycemia compared to non-diabetics and patients with properly controlled blood glucose levels.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Hyperglycemia resulting from Mtb infection was exacerbated early by sucrose treatment.
Random sampling of serum glucose values were compared to the mean glucose level of guinea pigs prior to sucrose feeding or infection (Pre-infection, n = 20) and served as the normal reference value. Serum glucose values of uninfected guinea pigs are similar to normal pre-infection values. Despite a mild increase in serum glucose associated with Mtb infection in the water-fed controls, significant exacerbation of hyperglycemia was only induced in sucrose-fed guinea pigs on day 30 of infection (n = 10). However,this difference was independent of sucrose feeding by day 60 (n = 9) of infection since persistent hyperglycemia was present in both sucrose- and water-fed groups. **p≤0.01.
Figure 2
Figure 2. Sucrose fed guinea pigs had significantly higher lung and extrapulmonary Mtb lesion burden.
The lung and lesion area was determined from hematoxylin and eosin stained tissue sections for each animal and the data expressed as mean percent involvement for each treatment group. The sucrose-fed guinea pigs had a significantly higher lesion burden compared to the water-fed control group in the lung (A) on day 60 of infection, spleen (B) on days 30 and 60 of infection, and mediastinal lymph node (C) on day 30 of infection. n = 10, *p≤0.05.
Figure 3
Figure 3. Sucrose feeding resulted in a more severe pulmonary lesion burden in Mtb infected guinea pigs.
Images represent the animals closest to the mean values for severity of lesion burden as determined by measuring lesion and normal lung area using morphometric analysis (see Figure 2). Pulmonary lesion severity was similar between water- (A) and sucrose-fed (B) guinea pigs at day 30 of infection but was more severe in the sucrose-fed animals by 60 days of infection (D) compared to the water-fed controls (C). Bar = 1000 µm. Hematoxylin and eosin stain. Insets: High magnification views of the TB lesions delineated on the subgross views of A–D; Bar = 100 µm.
Figure 4
Figure 4. Spleen lesions from extrapulmonary dissemination of Mtb were more severe and more numerous in sucrose-treated guinea pigs.
Images represent the animals closest to the mean severity of lesion burden as determined by measuring lesion and normal spleen area using morphometric analysis (see Figure 2). Splenic involvement was mild in the sucrose-fed guinea pigs at 30 days of infection (A) which was even less in the water-fed controls (B). However, splenic lesions were significantly more severe in the sucrose-fed animals by day 60 of infection (D) compared to the water-fed control group (C). Bar = 1000 µm. Hematoxylin and eosin stain. Insets: High magnification views of the TB lesions delineated on the subgross views of A–D; Bar = 100 µm.
Figure 5
Figure 5. Sucrose treatment resulted in higher bacterial burden in lung and spleen of Mtb infected guinea pigs.
Yield of viable bacilli from the lung was significantly higher in sucrose-fed guinea pigs on day 60 of infection compared to water-fed control animals. Even higher yields of bacilli were recovered from the spleens of sucrose-fed guinea pigs on day 60 of infection. No significant differences were observed in the mediastinal lymph nodes (LN). n = 10, *p≤0.05.
Figure 6
Figure 6. Elevated total serum free fatty acids occurred as a result of Mtb infection and not due to sucrose treatment.
Serum free fatty acids are similarly elevated between sucrose- (n = 9) and water-fed (n = 10) Mtb infected guinea pigs at day 30 of infection (A). The levels remain similarly elevated in sucrose- (n = 10) and water-fed (n = 10) Mtb infected guinea pigs at day 60 of infection with no increase in the mean values over time (B). Serum free fatty acid levels in sucrose-fed uninfected animals (day 30, n = 5; day 60, n = 5) are comparable to water-fed controls (day 30, n = 5; day 60, n = 4) at both time points. ***p≤0.001.
Figure 7
Figure 7. Mtb infection induces the formation and accumulation of AGEs in serum from guinea pigs independent of sucrose feeding.
Levels of serum AGEs were analyzed in sucrose- or water-fed guinea pigs with and without Mtb infection at both 30 and 60 days of infection. At 30 days of infection (A), mean serum AGEs are not elevated by either sucrose feeding or Mtb infection with levels remaining similar to uninfected controls. However, elevations in serum AGEs were present at 60 days due to Mtb infection (B) (sucrose-infected n = 10, water-infected n = 9) and were not exacerbated by sucrose feeding. AGE levels in uninfected, sucrose- and water-fed controls (n = 5 and n = 4, respectively) remain similar to levels at day 30. *p≤0.05.
Figure 8
Figure 8. Sucrose feeding of Mtb infected guinea pigs significantly increased tissue AGEs by day 30 of infection.
The mean score of AGE immunohistochemistry on lung is depicted in water- and sucrose-fed guinea pigs on days 30 and 60 of infection. Significantly increased AGE formation and accumulation was present within TB lesions by day 30 of infection in the sucrose-fed group compared to the water-fed controls. AGE levels at day 30 were similar to day 60 in the sucrose-fed group but additional AGEs accumulated within lesions by day 60 of the water-fed controls, which was not statistically significant. n = 10, *p≤0.05,***p≤0.001.
Figure 9
Figure 9. Sucrose feeding of Mtb infected guinea pigs increased lesion associated AGEs on day 30 and 60 of infection.
AGEs are evaluated by immunohistochemistry on lung tissue sections of Mtb infected guinea pigs. The majority of strong immunoreactivity was associated with TB lesions. Immunoreactivity was evident within the cytoplasm of macrophages forming granulomatous lesions and was also strongly present within central necrosis (N) of primary TB pulmonary lesions. (A) Minimal reactivity was confined to the serum, rare macrophages in the lesions (arrow) and minimally within areas of necrosis (N) of the water-fed control group at 30 days of infection. (B) In contrast, at 30 days of infection, the sucrose-fed animals had strong immunoreactivity in the majority of lesion macrophages (arrow) and in areas of necrosis (N). (C) By day 60 of infection, AGEs began to accumulate within macrophages (arrow) in the water-fed controls but minimal reactivity was present in areas of necrosis (N). (D) The sucrose-fed animals had strong immunoreactivity within the majority of macrophages (arrow) and in areas of necrosis (N) at day 60 of infection. All tissue sections depicted are representative of the mean AGE IHC score for each treatment group at each time point (see Figure 8). Bar = 100 µm.

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