Restoration of metabolic health by decreased consumption of branched-chain amino acids

doi:10.1113/JP275075

. 2018 Feb 15;596(4):623-645.

doi: 10.1113/JP275075. Epub 2017 Dec 27.

Restoration of metabolic health by decreased consumption of branched-chain amino acids

Nicole E Cummings^{1

2

3}, Elizabeth M Williams^{1

2}, Ildiko Kasza⁴, Elizabeth N Konon^{1

2}, Michael D Schaid^{1

2

5}, Brian A Schmidt^{1

2}, Chetan Poudel^{1

2}, Dawn S Sherman^{1

2}, Deyang Yu^{1

2

6}, Sebastian I Arriola Apelo^{1

2

7}, Sara E Cottrell^{1

2

8}, Gabriella Geiger^{1

2}, Macy E Barnes^{1

2}, Jaclyn A Wisinski^{1

2}, Rachel J Fenske^{1

2

5}, Kristina A Matkowskyj^{2

9

10}, Michelle E Kimple^{1

2

3

5

11}, Caroline M Alexander⁴, Matthew J Merrins^{1

2

12}, Dudley W Lamming^{1

2

3

5

6

9}

Affiliations

¹ Department of Medicine, University of Wisconsin-Madison, Madison, WI, USA.
² William S. Middleton Memorial Veterans Hospital, Madison, WI, USA.
³ Endocrinology and Reproductive Physiology Graduate Training Program, University of Wisconsin-Madison, Madison, WI, USA.
⁴ McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI, USA.
⁵ Interdisciplinary Graduate Program in Nutritional Sciences, University of Wisconsin-Madison, Madison, WI, USA.
⁶ Molecular and Environmental Toxicology Program, University of Wisconsin-Madison, Madison, WI, USA.
⁷ Department of Dairy Science, University of Wisconsin-Madison, Madison, WI, USA.
⁸ Rural and Urban Scholars in Community Health Program, University of Wisconsin-Madison, Madison, WI, USA.
⁹ University of Wisconsin Carbone Cancer Center, Madison, WI, USA.
¹⁰ Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, WI, USA.
¹¹ Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, WI, USA.
¹² Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, USA.

PMID: 29266268
PMCID: PMC5813603
DOI: 10.1113/JP275075

Restoration of metabolic health by decreased consumption of branched-chain amino acids

Nicole E Cummings et al. J Physiol. 2018.

. 2018 Feb 15;596(4):623-645.

doi: 10.1113/JP275075. Epub 2017 Dec 27.

Authors

Affiliations

¹ Department of Medicine, University of Wisconsin-Madison, Madison, WI, USA.
² William S. Middleton Memorial Veterans Hospital, Madison, WI, USA.
³ Endocrinology and Reproductive Physiology Graduate Training Program, University of Wisconsin-Madison, Madison, WI, USA.
⁴ McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI, USA.
⁵ Interdisciplinary Graduate Program in Nutritional Sciences, University of Wisconsin-Madison, Madison, WI, USA.
⁶ Molecular and Environmental Toxicology Program, University of Wisconsin-Madison, Madison, WI, USA.
⁷ Department of Dairy Science, University of Wisconsin-Madison, Madison, WI, USA.
⁸ Rural and Urban Scholars in Community Health Program, University of Wisconsin-Madison, Madison, WI, USA.
⁹ University of Wisconsin Carbone Cancer Center, Madison, WI, USA.
¹⁰ Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, WI, USA.
¹¹ Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, WI, USA.
¹² Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, USA.

PMID: 29266268
PMCID: PMC5813603
DOI: 10.1113/JP275075

Abstract

Key points: We recently found that feeding healthy mice a diet with reduced levels of branched-chain amino acids (BCAAs), which are associated with insulin resistance in both humans and rodents, modestly improves glucose tolerance and slows fat mass gain. In the present study, we show that a reduced BCAA diet promotes rapid fat mass loss without calorie restriction in obese mice. Selective reduction of dietary BCAAs also restores glucose tolerance and insulin sensitivity to obese mice, even as they continue to consume a high-fat, high-sugar diet. A low BCAA diet transiently induces FGF21 (fibroblast growth factor 21) and increases energy expenditure. We suggest that dietary protein quality (i.e. the precise macronutrient composition of dietary protein) may impact the effectiveness of weight loss diets.

Abstract: Obesity and diabetes are increasing problems around the world, and although even moderate weight loss can improve metabolic health, reduced calorie diets are notoriously difficult to sustain. Branched-chain amino acids (BCAAs; leucine, isoleucine and valine) are elevated in the blood of obese, insulin-resistant humans and rodents. We recently demonstrated that specifically reducing dietary levels of BCAAs has beneficial effects on the metabolic health of young, growing mice, improving glucose tolerance and modestly slowing fat mass gain. In the present study, we examine the hypothesis that reducing dietary BCAAs will promote weight loss, reduce adiposity, and improve blood glucose control in diet-induced obese mice with pre-existing metabolic syndrome. We find that specifically reducing dietary BCAAs rapidly reverses diet-induced obesity and improves glucoregulatory control in diet-induced obese mice. Most dramatically, mice eating an otherwise unhealthy high-calorie, high-sugar Western diet with reduced levels of BCAAs lost weight and fat mass rapidly until regaining a normal weight. Importantly, this normalization of weight was mediated not by caloric restriction or increased activity, but by increased energy expenditure, and was accompanied by a transient induction of the energy balance regulating hormone FGF21 (fibroblast growth factor 21). Consumption of a Western diet reduced in BCAAs was also accompanied by a dramatic improvement in glucose tolerance and insulin resistance. Our results link dietary BCAAs with the regulation of metabolic health and energy balance in obese animals, and suggest that specifically reducing dietary BCAAs may represent a highly translatable option for the treatment of obesity and insulin resistance.

Keywords: branched-chain amino acids; diabetes; obesity; protein restriction.

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Figures

**Figure 1. Normal calorie diets with reduced levels of BCAAs promote rapid weight loss**
A, schematic representation of the experimental plan; mice were preconditioned with a WD for 12 weeks and then randomized to the five experimental groups shown, whereas chow‐fed Control mice were placed on an amino acid defined Control diet. B, weight as well as (C) adipose and lean mass, of mice in each experimental group, was tracked (n = 12 mice per group). D, epididymal WAT was collected at necropsy and weighed (n = 11–12 mice per group; ^* P < 0.05, Dunnett's test following ANOVA, ^** P < 0.05, Bonferroni's test). E, 2 and 10 weeks following the start of the specified dietary intervention, food intake was assessed over a 4 day period in home cages, calculated as kcal day g⁻¹ body weight (n = 6–7 cages per group; means with the same lowercase letter are not significantly different from each other, Tukey–Kramer test following ANOVA, P < 0.05). F, spontaneous activity and energy expenditure were measured using metabolic chambers ∼7–8 weeks after the start of the dietary intervention (n = 5–8 mice per group; means with the same lowercase letter are not significantly different from each other, Tukey–Kramer test following ANOVA, P < 0.05). Error bars represent the SE.

**Figure 2. Consumption of an ExLow BCAA diet reduces adipose tissue and reverses diet‐induced hepatic steatosis**
A, paraffin‐embedded skin samples were collected at necropsy ∼15 weeks following the start of the specified dietary intervention, sectioned, and H&E stained, and the thickness of dermal WAT was quantified (B) for non‐anagen stage skin samples, measuring from muscle to dermis; scale bar = 200 μm (n = 5–7 mice per group, ^* P < 0.05, Dunnett's test following ANOVA, ^** P < 0.05, Bonferroni's test). C, liver samples were stained with Oil‐Red‐O and (D) droplet size was quantified; scale bar = 200 μm (n = 3 mice per group, ^* P < 0.05 vs. WD, Dunnett's test following ANOVA). E, lipogenic gene expression was measured in the livers of fasted mice (n = 5–6 mice per group, ^* P < 0.05 vs. WD, Dunnett's test following two‐way repeated measures ANOVA). Error bars represent the SE.

**Figure 3. Consumption of BCAAs inversely correlates with glucose tolerance and insulin sensitivity**
Glucose (A) and (B) insulin tolerance tests were conducted at the specified times after the start of the dietary interventions (n = 10–12 per group; for the area under the curve, means with the same lowercase letter are not significantly different from each other, Tukey–Kramer test following ANOVA, P < 0.05). C–E, mice were fasted overnight and (C) blood glucose and (D) insulin were measured and (E) the HOMA2‐IR was calculated after 5 weeks on the specified diets (n = 5–7 mice per group; means with the same lowercase letter are not significantly different from each other, Tukey–Kramer test following ANOVA, P < 0.05). Error bars represent the SE.

**Figure 4. Specifically reducing dietary BCAAs in the context of a WD promotes rapid weight loss and reduced adiposity**
A, schematic representation of experimental plan; mice were preconditioned with a WD for 12 weeks and then randomized to the four experimental groups shown, whereas chow‐fed Control mice were placed on an amino acid defined Control diet. B, weight, as well as (C) adipose and lean mass of mice in each experimental group was tracked (n = 12 mice per group). D, food intake was measured 2 weeks after special diet feeding began (n = 8 cages per group; means with the same lowercase letter are not significantly different from each other, Tukey–Kramer test following ANOVA, P < 0.05). E, body composition at diet intervention start and 12 weeks later. Error bars represent the SE.

**Figure 5. Specifically reducing dietary BCAAs in the context of a WD reduces adipose tissue and reverses diet‐induced hepatic steatosis**
A, skin samples were collected after feeding mice the indicated diets for ∼14 weeks, sectioned, H&E stained and the thickness of dermal WAT was quantified for non‐anagen stage skin samples, measuring from muscle to dermis; scale bar = 200 μm (n = 6 mice per group, ^* P < 0.05 vs. WD Control AA, Dunnett's test following ANOVA). B, OCT‐embedded liver samples were cryosectioned and stained with Oil‐Red‐O, and droplet size was quantified; scale bar = 200 μm (n = 3 mice per group, ^* P < 0.05 vs. WD Control AA, Dunnett's test following ANOVA). C, triglyceride levels in liver as mg dL^–1 mg^–1 of tissue assayed (n = 6 mice per group, ^* P < 0.05 vs. WD Control AA, Dunnett's test following ANOVA). D, lipogenic gene expression in the livers of mice on the specified diets was measured by quantitative PCR after an overnight fast (n = 8–9 mice per group, ^* P < 0.05, #P < 0.1 vs. WD Control AA, Dunnett's test following ANOVA). Error bars represent the SE.

**Figure 6. Reduction of dietary BCAAs improves glycaemic control even in mice continuing to consume a high‐calorie, high‐fat, high‐sugar WD**
Glucose (A) and (B) insulin tolerance tests were conducted 3 and 4 weeks, respectively, after the start of the dietary interventions (n = 12–16 per group; for the area under the curve, means with the same lowercase letter are not significantly different from each other, Tukey–Kramer test following ANOVA, P < 0.05). C–E, fasting (C) blood glucose and (D) insulin were measured and (E) the HOMA2‐IR was calculated after 5 weeks on the specified diets (n = 3–7 mice per group; ^* P < 0.05, #P < 0.12 vs. WD Control AA, Dunnett's test following ANOVA).

**Figure 7. *Ex vivo* analysis of the effect of altering dietary BCAAs on pancreatic beta cells**
A, an *ex vivo* insulin secretion assay was performed to assess (left) insulin secretion per islet and (right) islet insulin content in response to low (1.7 mm) and high (16.7 mm) glucose in mice kept on the indicated diets for ∼14 weeks (n = 6 mice per group, ^* P < 0.05 vs. WD Control AA, Dunnett's test following ANOVA). B, the mitochondrial membrane potential was measured in *ex vivo* isolated pancreatic islets stimulated with low (2 mm) and high (20 mm) glucose levels (n = 44–74 islets per group; ^* P < 0.05 vs. WD Control AA, Dunnett's test following ANOVA). Error bars represent the SE.

**Figure 8. Metabolic impact of sustained consumption of diets with altered dietary BCAAs**
A, food intake over a 24 h period (n = 2–5 mice per group; means with the same lowercase letter are not significantly different from each other, Bonferroni test, P < 0.05). B, RER, (C) spontaneous activity and (D) energy expenditure were measured ∼12 weeks after the start of the dietary intervention (n = 4–5 mice per group; means with the same lowercase letter are not significantly different from each other, Tukey–Kramer test following ANOVA, P < 0.05). E, FGF21 was measured in the plasma of mice following an overnight fast (n = 4 mice per group; means with the same lowercase letter are not significantly different from each other, Tukey–Kramer test following ANOVA, P < 0.05). F, *Fgf21* gene expression in the liver of mice following an overnight fast was assessed by quantitative PCR (n = 8–9 mice per group; #P < 0.1 vs. WD Control AA, Dunnett's test following ANOVA). Error bars represent the SE.

**Figure 9. A Western reduced BCAA diet transiently induces FGF21 and increases energy expenditure**
A, weight of DIO mice switched to the indicated diet at time 0 (n = 8 per group). B, change in fat and lean mass of mice placed on each diet for 7 days (n = 8 mice per group, ^* P < 0.05 vs. WD Control AA, Dunnett's test following ANOVA). C, RER, spontaneous activity and energy expenditure were measured 1 week after the diet switch (n = 6–8 mice per group, ^* P < 0.05 vs. WD Control AA, Dunnett's test following ANOVA). D, energy expenditure over the course of a 24 h cycle starting at ∼10.00 h (n = 8 mice per group; dark outline of symbol indicates P < 0.05 vs. WD Control AA (Dunnett's test following two‐way repeated measures ANOVA). E, FGF21 was measured in the plasma of mice following an overnight fast (n = 6 mice per group; means with the same lowercase letter are not significantly different from each other, Tukey–Kramer test following ANOVA, P < 0.05). Error bars represent the SE.

**Figure 10. A Western reduced BCAA diet does not beige WAT but activates BAT**
Following an overnight fast, tissues were collected from DIO mice switched to the indicated diets for 12 days. A, inguinal and gonadal WAT (iWAT and gWAT, respectively) depots were collected, sectioned, and H&E stained; scale bar = 200 μm. B, the expression of UCP1 in iWAT and gWAT was determined by Western blotting. C, UCP1 expression in iWAT and gWAT was quantified relative to HSP90 (n = 8 mice per group, ^* P < 0.05 vs. WD Control AA, Dunnett's test following ANOVA). D, *Ucp1* gene expression in BAT was assessed by quantitative PCR (n = 6 mice per group, ^* P < 0.05 vs. WD Control AA, Dunnett's test following ANOVA). E, BAT was collected, sectioned, and H&E stained, and lipid droplet size was quantified; scale bar = 200 μm (n = 6 mice per group; means with the same lowercase letter are not significantly different from each other, Tukey–Kramer test following ANOVA, P < 0.05). F, *Bmp8* gene expression in BAT was assessed by quantitative PCR (n = 6 mice per group; ^* P < 0.05 vs. WD Control AA, Dunnett's test following ANOVA). Error bars represent the SE.

**Figure 11. Specifically reducing dietary BCAAs or all AAs restores metabolic health to DIO mice**
Altering dietary levels of either the BCAAs or all AAs promotes leanness and restores blood glucose control to DIO mice, but these two dietary interventions have distinct effects on energy expenditure (EE), food intake and blood levels of FGF21. Further, these metabolic effects vary between an acute phase (an ∼4 week long period following the diet switch characterized by rapid weight loss), and a chronic phase that persists once mice have reached a stable weight.

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Comment in

Dietary branched chain amino acids and metabolic health: when less is more.
Hill CM, Morrison CD. Hill CM, et al. J Physiol. 2018 Feb 15;596(4):555-556. doi: 10.1113/JP275613. Epub 2018 Jan 19. J Physiol. 2018. PMID: 29313984 Free PMC article. No abstract available.
Restricting branched-chain amino acids: an approach to improve metabolic health.
Anderson JG, Hintze K, Marchant ED. Anderson JG, et al. J Physiol. 2018 Jul;596(13):2469-2470. doi: 10.1113/JP276205. Epub 2018 May 30. J Physiol. 2018. PMID: 29718534 Free PMC article. No abstract available.
Reducing branched-chain amino acid intake to reverse metabolic complications in obesity and type 2 diabetes.
Yadao DR, MacKenzie S, Bergdahl A. Yadao DR, et al. J Physiol. 2018 Aug;596(16):3455-3456. doi: 10.1113/JP276274. Epub 2018 Jun 21. J Physiol. 2018. PMID: 29791751 Free PMC article. No abstract available.

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References

1. Ananieva EA, Van Horn CG, Jones MR & Hutson SM (2017). Liver BCATm transgenic mouse model reveals the important role of the liver in maintaining BCAA homeostasis. J Nutr Biochem 40, 132–140. - PMC - PubMed
1. Arriola Apelo SI, Neuman JC, Baar EL, Syed FA, Cummings NE, Brar HK, Pumper CP, Kimple ME & Lamming DW (2016). Alternative rapamycin treatment regimens mitigate the impact of rapamycin on glucose homeostasis and the immune system. Aging Cell 15, 28–38. - PMC - PubMed
1. Baar EL, Carbajal KA, Ong IM & Lamming DW (2016). Sex‐ and tissue‐specific changes in mTOR signaling with age in C57BL/6J mice. Aging Cell 15, 155–166. - PMC - PubMed
1. Barb D, Portillo‐Sanchez P & Cusi K (2016). Pharmacological management of nonalcoholic fatty liver disease. Metabolism 65, 1183–1195. - PubMed
1. Batch BC, Shah SH, Newgard CB, Turer CB, Haynes C, Bain JR, Muehlbauer M, Patel MJ, Stevens RD, Appel LJ, Newby LK & Svetkey LP (2013). Branched chain amino acids are novel biomarkers for discrimination of metabolic wellness. Metabolism 62, 961–969. - PMC - PubMed

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[1] Ananieva EA, Van Horn CG, Jones MR & Hutson SM (2017). Liver BCATm transgenic mouse model reveals the important role of the liver in maintaining BCAA homeostasis. J Nutr Biochem 40, 132–140. - PMC - PubMed

[2] Ananieva EA, Van Horn CG, Jones MR & Hutson SM (2017). Liver BCATm transgenic mouse model reveals the important role of the liver in maintaining BCAA homeostasis. J Nutr Biochem 40, 132–140. - PMC - PubMed

[3] Arriola Apelo SI, Neuman JC, Baar EL, Syed FA, Cummings NE, Brar HK, Pumper CP, Kimple ME & Lamming DW (2016). Alternative rapamycin treatment regimens mitigate the impact of rapamycin on glucose homeostasis and the immune system. Aging Cell 15, 28–38. - PMC - PubMed

[4] Arriola Apelo SI, Neuman JC, Baar EL, Syed FA, Cummings NE, Brar HK, Pumper CP, Kimple ME & Lamming DW (2016). Alternative rapamycin treatment regimens mitigate the impact of rapamycin on glucose homeostasis and the immune system. Aging Cell 15, 28–38. - PMC - PubMed

[5] Baar EL, Carbajal KA, Ong IM & Lamming DW (2016). Sex‐ and tissue‐specific changes in mTOR signaling with age in C57BL/6J mice. Aging Cell 15, 155–166. - PMC - PubMed

[6] Baar EL, Carbajal KA, Ong IM & Lamming DW (2016). Sex‐ and tissue‐specific changes in mTOR signaling with age in C57BL/6J mice. Aging Cell 15, 155–166. - PMC - PubMed

[7] Barb D, Portillo‐Sanchez P & Cusi K (2016). Pharmacological management of nonalcoholic fatty liver disease. Metabolism 65, 1183–1195. - PubMed

[8] Barb D, Portillo‐Sanchez P & Cusi K (2016). Pharmacological management of nonalcoholic fatty liver disease. Metabolism 65, 1183–1195. - PubMed

[9] Batch BC, Shah SH, Newgard CB, Turer CB, Haynes C, Bain JR, Muehlbauer M, Patel MJ, Stevens RD, Appel LJ, Newby LK & Svetkey LP (2013). Branched chain amino acids are novel biomarkers for discrimination of metabolic wellness. Metabolism 62, 961–969. - PMC - PubMed

[10] Batch BC, Shah SH, Newgard CB, Turer CB, Haynes C, Bain JR, Muehlbauer M, Patel MJ, Stevens RD, Appel LJ, Newby LK & Svetkey LP (2013). Branched chain amino acids are novel biomarkers for discrimination of metabolic wellness. Metabolism 62, 961–969. - PMC - PubMed

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Restoration of metabolic health by decreased consumption of branched-chain amino acids

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Restoration of metabolic health by decreased consumption of branched-chain amino acids

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