Department of Endocrinology St Vincent’s Hospital, Sydney Bile acids more closely align with insulin resistance and visceral and hepatic adiposity than total body fat in adult humans Ramy Bishay Department of Endocrinology St Vincent’s Hospital, Sydney

Bile Acid Physiology 7a-hydroxylase (CYP7A1) Produced exclusively in the liver as end-products of cholesterol catabolism Facilitate hepatobiliary secretion (endogenous metabolites, xenobiotics) Intestinal absorption lipophilic nutrients (ADEK) Energy expenditure Glucose metabolism 7a) Classical pathway (90% contribution) vs alternative (acidic pathway) 7a hydroxylase – first and most important, rate-limiting step in primary BA synthesis CA vs CDCA dictated by enzymatic reaction of sterol 12α-hydroxylase (CYB8B1) -> controls the synthesis of cholic acid and as such is under tight transcriptional control CDCA (45%), CA (31%) Diab Care 32(2): 2009; J of Lipids, Vol 2012, Article ID 754067; Pharmacol Rev 66:948–983, Oct 2014; Michael W King, 1996–2016 themedicalbiochemistrypage.org; J of Lipids, Vol 2012, Article ID 754067

Bile Acid Physiology Homeostasis self-governed Modulated by multiple receptors at different tissues FXR, TGR5, RXR, LXR 50% cholesterol turnover Prevent crystallisation and gallstones by solubilising cholesterol BA are physiological ligands for FXR

Study Aims 1. To determine if bile acid levels are increased in humans with diabetes In vitro: Hyperglycaemia stimulates 7a-hydroxylation (increased bile acid synthesis) Mouse models (Type 1 and Type 2 diabetes): Hyperglycaemia increased bile acid synthesis Larger bile acid pool size in db/db mice (Cholic, Deoxycholic acids) Humans: Bile acid sequestration improves hyperglycaemia (HbA1c -0.5%), insulin sensitivity (via GLP-1) JCEM 98, E708-E712; Cell Metab, 10, 167-177; Biochem Biophys Res Commun, 329, 386-390; Sci Rep 2, 430; Nature, vol. 439, no. 7075, pp. 484–489, 2006; Eur J Endocrinol. 2014 Aug;171(2):R47-65; Ann Intern Med 1994;121:416–422; Curr. Opin. Lipidol. 23, 43-55; Trends Endocinol Metab 2014, Vol. 25, No. 5 ; Hepatology 52, 1455–1464; Diabetes 62, 4184-4191; Diabetes Care Vol 32, supp 2, 2009.

Study Aims 2. To determine if bile acid levels are associated with obesity Higher fasting bile acids (cholic acid) in obese vs non-obese Weight loss Bile acid sequestration Activation of TGR5 (increased E expenditure) Promote GLP-1 (certain bile acids, via TGR5) Insulin reduced serum bile acid but affect blunted in obese subjects Roux-en-Y gastric bypass (but not lap-banding) result in elevated circulating bile acids 3. To determine if bile acid levels are increased in obese humans independent of insulin resistance? ??? TGR5 - GPCR expressed in intestine, BAT, WAT, gall bladder less so in liver, muscle BA activation of TGR5 - In brown adipose tissue -> induces D2, FA oxidation genes transcription J Clin Endocrinol Metab 101: 1935–1944, 2016; Trends Endocinol Metab 2014, Vol. 25, No. 5; Hepatology 52, 1455–1464; Diabetes 62, 4184-4191; Diabetes Care Vol 32, supp 2, 2009.

Methods 71 adult volunteers 4 groups based on BMI, homeostatic model assessment of insulin resistance (HOMA-IR) and a 75-g OGTT: 1. Lean insulin-sensitive (BMI≤25 kg/m2, HOMA-IR<2.0, n=19) 2. Overweight/obese insulin-sensitive non-diabetic (Obsen, BMI>25 kg/m2, HOMA-IR<1.5, n=11) 3. Overweight/obese insulin-resistant (Obres, BMI>25 kg/m2, HOMA-IR>3.0, n=20) 4. Type 2 diabetes mellitus (T2DM, n=21). Insulin sensitivity measured by hyperinsulinaemic (80 mUm−2min−1)–euglycaemic (5 mmol/l) clamp Body composition and central abdominal fat by DXA Visceral fat area by CT Fasting insulin, adiponectin, FGF21 BA (baseline and during clamp) Primary – CA, CDCA, Secondary – DCA Conjugated

Baseline Characteristics

Results: Total Bile Acids

Chenodeoxycholic Acid Results: 1° Bile Acids Cholic Acid Chenodeoxycholic Acid

Results: 2° & Conjugated Bile Acids Deoxycholic Acid Conjugated BA

- + Correlations Cholic (1° BA) Chenodeoxycholic Deoxycholic (2° BA) Conjugated BA Waist Circumference BMI Central fat (% or kg) Visceral fat (cm2) P = 0.06 + Liver fat Insulin P = 0.051 GDR/FFM P = 0.054 - FFM FGF 21 Adiponectin + -

Summary Bile acids are lower in insulin sensitive individuals, independent of adiposity Obsens have significantly lower: Total and conjugated bile acids vs T2D Total bile acids and chenodeoxycholic acid (1° BA) vs Obres Lean have lower Deoxycholic acid (2° BA) compared with T2D Bile acid levels are suppressed with insulin Bile acids are associated with metabolic risk factors Cholic acid (1°): central/visceral adiposity, hepatic fat and insulin resistance Deoxycholic acid (2°): type 2 diabetes, central/visceral fat, adiponectin, elevated fasting insulin

Conclusions Bile acids are complex signalling molecules Align closely to insulin resistance May have a pathophysiological role in the development of insulin resistance, obesity and hepatic adiposity Supports previous positive studies on therapeutic role of bile acid sequestration and signalling blockade on glycaemic control (mixed results)

Thank you Acknowledgements Garvan Institute of Medical Research & St Vincent’s Hospital, Dept Endocrinology Dr Katherine Tonks A/Prof Jerry Greenfield Prof Don Chisholm Dr Dorit Samocha-Bonet The Charles Perkins Centre, University of Sydney Prof David E James Storr Liver Centre, Westmead Millennium Institute and Westmead Hospital Prof Jacob George