2. PREVALENCE OF NAFLD IN DIABETIC PATIENTS  The world-wide prevalence of non-alcoholic fatty liver disease (NAFLD) is: o Western countries: 15% to 40% o Asian countries: 9% to 40%  NAFLD is emerging as an important cause of liver disease in India  In general Indian population, epidemiological studies have suggested the prevalence of NAFLD to be around 9% to 32%  Higher incidence has been reported in Indian patients who are overweight/obese and diabetic/prediabetic Kalra S, et al. J Assoc Physicians India. 2013 Jul;61(7):448-53.

3. PREVALENCE OF NAFLD IN DIABETES PATIENTS  A study by Kalra S et al., in the year 2013, studied frequency and risk factors of NAFLD in non-alcoholic Indian type 2 diabetic (T2DM) patients o Prevalence of NAFLD in patients with T2DM was 56.5% across India. o Prevalence of NAFLD ranged from 44.1% in western India to 72.4% in northern states (Table 1) o Highest prevalence was reported in age group of 61  70 years (61.8%) Kalra S, et al. J Assoc Physicians India. 2013 Jul;61(7):448-53.

4. o Greater prevalence was seen in females (60%) as compared to males (54.3%) T2DM patients (Table 2) Kalra S, et al. J Assoc Physicians India. 2013 Jul;61(7):448-53. PREVALENCE OF NAFLD IN DIABETES PATIENTS

5.  NAFLD is associated with several conditions, including: 1 o Obesity (69  100%) o Diabetes mellitus (36  5%) o Hyperlipidemia (20  81%)  Patients with T2DM and co-morbid conditions like obesity, hypertension and dyslipidemia reported greater risk for NAFLD by 14%, 17% and 38%, respectively 2  Diabetes mellitus is reported in about 75% of patients with non-alcoholic steatohepatitis (NASH) 1  On ultrasonography, more likelihood of fatty liver changes (up to 70%) in obese, middle-aged females with DM was noted 1 1.Dabhi AS et al. JIACM. 2008;9(1):36-41. 2.Kalra S, et al. J Assoc Physicians India. 2013 Jul;61(7):448-53. NAFLD AND ASSOCIATED CONDITIONS

6. Dabhi AS et al. JIACM. 2008;9(1):36-41. PATHOGENESIS AND NATURAL HISTORY OF NAFLD

7.  Epidemiological studies in patients with diabetes suggest an increased risk of chronic liver disease, cirrhosis, hepatocellular carcinoma and increased mortality due to hepatic disease  There is an increased risk of developing hepatic morbidity among patients with NAFLD  NAFLD is the most common liver disease seen in diabetic patients, and it has been reported that 20% to 50% patients with NAFLD have diabetes  DM and NAFLD demonstrate a common pathogenic link i.e., insulin resistance, both hepatic as well as peripheral  Type 2 diabetes, obesity, older age and metabolic syndrome are the risk factors for rapid progression of NAFLD Amarapurkar D. Medicine Update. 2010;20:466-469. PATHOGENIC LINK BETWEEN NAFLD AND DIABETES MELLITUS

8.  Diabetes development may result due to NAFLD, which accounts for increase in glucose production in the liver and exacerbation of hepatic and systemic insulin resistance possibly via systemic release of pro-inflammatory factors and some liver-secreted proteins with diabetogenic properties, such as fetuin-A, FGF-21 and RBP-4 (Figure 2) Anstee QM et al. Nature Reviews Gastroenterology & Hepatology. 2013;10:330-344. PATHOGENIC LINK BETWEEN NAFLD AND DIABETES MELLITUS

9.  A close association between the presence of diabetes mellitus and NAFLD has been reported in several studies  Delivery of insulin directly to the portal vein after secretion via a similar route as absorbed glucose, and elimination of large portion of portal insulin at the first pass may a suggest significant link between T2DM, insulin resistance (IR) and NAFLD  Presence of obesity in NAFLD may be associated with dysfunctional adipose tissue, and lipotoxicity that may lead to insulin resistance and pancreatic β-cell dysfunction Firneisz G. World J Gastroenterol.2014 July 21; 20(27): 9072-9089. PATHOGENIC LINK BETWEEN NAFLD AND DIABETES MELLITUS

10.  A study conducted by Prashanth M et al., in 204 Indian T2DM patients, showed hepatic fatty infiltration in 62.2% of the patients, and confirmed NAFLD by liver biopsy with 54.11% prevalence in T2DM patients (Figure 3)  Steatohepatitis and fibrosis were reported in 38.9% and 23.2% of T2DM patients, respectively  A study by Leite et al., reported 78% prevalence of NASH on histological evaluation among 100 patients with T2DM and also demonstrated ultrasonography evidence of NAFLD Firneisz G. World J Gastroenterol 2014 July 21; 20(27): 9072-9089. PATHOGENIC LINK BETWEEN NAFLD AND DIABETES MELLITUS

11.  Chronic inflammatory disease such as NAFLD demonstrated lower vitamin D levels suggesting pleiotropic role in the pathogenesis of NAFLD  Insulin resistance, metabolic syndrome and proinflammatory cytokines have been associated in the development and progression of NAFLD  Negative correlation has been reported between serum levels of vitamin D and cases of insulin resistance and metabolic syndrome  Reduction in insulin resistance has been reported with vitamin D supplementation among obese children  Reduced levels of vitamin D levels are independently associated with increased severity of steatosis, necroinflammation, and fibrosis in NAFLD (Figure 4) Yin K et al. Journal of Inflammation Research. 2014:7 69–87. CLINICAL LINK BETWEEN VITAMIN D AND NAFLD

12. Yin K et al. Journal of Inflammation Research.2014:7 69–87. CLINICAL LINK BETWEEN VITAMIN D AND NAFLD  It has been reported in several NAFLD patients that steatosis follows inflammation  Therefore, ability of vitamin D to modulate the immune/inflammation system has been reported to be associated with the development of NAFLD

13. Vitamin D signaling  Intracellular signals of vitamin D are mediated through its vitamin D receptor that is expressed in the liver  Over 200 genes associated with glucose and lipid metabolism, inflammation, cellular proliferation and differentiation and apoptosis are regulated by VDR  Four single nucleotide polymorphisms (SNPs) demonstrated significant association with NAFLD in a GWAS study performed in NAFLD subjects  Out of these four SNPs, GC gene was predominately expressed in hepatocytes, and was responsible for coding of vitamin D binding protein, which has been regarded as an important carrier protein for vitamin D Eliades M, et al. World J Gastroenterol.2015; 21(6): 1718-1727. MECHANISMS LINKING VITAMIN D DEFICIENCY TO NAFLD

14. Role of vitamin D in adipose tissue, insulin resistance and hepatic inflammation  In NAFLD, insulin resistance is associated with the development of oxidative stress and lipotoxicity  Accumulation of free fatty acids causing hepatic steatosis is associated with chronic hepatic inflammation  Human studies have suggested significant correlation between increased cytokine gene expression and histological severity in the liver of patients with NASH as compared to obese controls with normal liver  Further, reduced adipocyte production of adiponectin has demonstrated an essential role in the pathogenesis of NAFLD progression Eliades M, et al. World J Gastroenterol.2015; 21(6): 1718-1727. MECHANISMS LINKING VITAMIN D DEFICIENCY TO NAFLD

15. Role of vitamin D in Adipose tissue, insulin resistance and hepatic inflammation  The role of vitamin D in adipokine activity has been elucidated in several studies  A study by Vaidya et al., demonstrated a positive link between levels of adiponectin and 25(OH)D concentrations in a large cohort of patients  A double-blind, randomised, controlled trial of Iranian type 2 diabetic patients, resulted in a significant increase of serum adipokines including adiponectin and decreased cellular secretion of the inflammatory cytokines IL-6 and IL-1β with daily intake of 1000 IU vitamin D either with or without extra calcium for 12 weeks  Importantly, preliminary studies have suggested that vitamin D deficiency, associated with upregulation of hepatic inflammatory and oxidative stress genes may be responsible for worsening of NAFLD. Supplementation with vitamin D have reported to be effective in reduction adipose tissue inflammation (Figure 5) Eliades M, et al. World J Gastroenterol.2015; 21(6): 1718-1727. MECHANISMS LINKING VITAMIN D DEFICIENCY TO NAFLD

16. Eliades M, et al. World J Gastroenterol.2015; 21(6): 1718-1727. MECHANISMS LINKING VITAMIN D DEFICIENCY TO NAFLD

17. Role of vitamin D deficiency and intestinal microbiome and innate immunity in NAFLD  In a study conducted in Italy, patients with NAFLD have demonstrated increased gut permeability and small intestinal bacterial overgrowth (SIBO), which suggested correlation with the severity of steatosis, but not with presence of NASH  Vitamin D deficiency may cause activation of toll-like receptor-2 (TLR2) and TLR4 by way of CD14/LBP, and stimulation of downstream inflammatory signaling molecules that lead to steatosis and inflammation, which may be responsible for development and progression of NAFLD  However, role of vitamin D in innate immunity is still unclear Eliades M, et al. World J Gastroenterol.2015; 21(6): 1718-1727. MECHANISMS LINKING VITAMIN D DEFICIENCY TO NAFLD

18. Vitamin D and hepatic fibrosis  The development of liver fibrosis in NAFLD is considered as strongest predictor for disease-specific mortality  Fibrosis may lead to liver dysfunction by causing accumulation of extracellular matrix (ECM) with subsequent destruction of the normal liver architecture  Hepatic stellate cells (HSCs) are responsible for excessive deposition of ECM proteins, predominantly type I collagen, and hence, HSCs play a critical role in the development of liver fibrosis Eliades M, et al. World J Gastroenterol.2015; 21(6): 1718-1727. MECHANISMS LINKING VITAMIN D DEFICIENCY TO NAFLD

19. A study by Nwosu BU et al., was conducted to determine the link between mild hepatic dysfunction and impaired 25-hydroxylation of pre-vitamin D, and vitamin D deficiency and its effect on glycaemic control in children with T1DM (n=81) and T2DM (n=40).  47.5% patients with T2DM reported vitamin D deficiency [25(OH)D<15 ng/mL]  Elevated levels of transaminases were reported in T2DM patients compared to T1DM patients (AST 39.3  2.0 vs. 22.4  1.4, p<0.001; and ALT 30.6  1.8 vs. 18.7  1.3, p<0.001)  Significant inverse linear relation between 25(OH)D and HbA 1c levels was reported in T2DM patients (  = –0.40, p=0.011) [Figure 6] Nwosu BU et al. J Pediatr Endocrinol Metab. 2012;25(1-2)181-6. CLINICAL EVIDENCE ON LOW LEVELS OF VITAMIN D IN DIABETIC PATIENTS WITH NAFLD

20.  Also, significant inverse relation between 25(OH)D and ALT was reported in T2DM patients (β= –0.40, p=0.02) [Figure 7]  No significant change was reported between 25(OH)D and AST in T2DM patients (β= –0.28, p=0.10)  No relation between 25(OH)D and ALT (β=0.06, p=0.77) and between 25(OH)D and AST (β=0.09, p=0.65) was observed in T1DM patients  Therefore, study results suggested that hepatic dysfunction is associated with vitamin D deficiency, which further affects glycaemic control Nwosu BU et al. J Pediatr Endocrinol Metab. 2012;25(1-2)181-6. CLINICAL EVIDENCE ON LOW LEVELS OF VITAMIN D IN DIABETIC PATIENTS WITH NAFLD

21.  In a study by Targher G et al., link between serum 25-hydroxyvitamin D3 [25(OH)D] concentrations and liver histology in patients with NAFLD was evaluated.  This study included 60 patients with NAFLD and 60 healthy controls.  Compared to controls, NAFLD patients reported marked reduction in serum 25(OH)D levels (51.0 ± 22 vs. 74.5 ± 15 nmol/L, p<0.001)  Also, the study reported that decreased 25(OH)D concentrations were closely associated with the histological severity of hepatic steatosis, necroinflammation and fibrosis (p<0.001)  Therefore, study results have suggested that status of vitamin D deficiency may be responsible for the development and progression of NAFLD Targher G et al. Nutrition, Metabolism & Cardiovascular Diseases. 2007;17:517-524. CLINICAL EVIDENCE ON LOW LEVELS OF VITAMIN D IN DIABETIC PATIENTS WITH NAFLD

22.  A meta-analysis was also conducted by Eliades M et al., to review the relation between NAFLD and vitamin D levels  A total of 17 studies were evaluated, out of which 9 studies were used for quantitative analysis  The study results demonstrated that NAFLD patients had 0.36 ng/mL lower levels of vitamin D [25(OH)D levels] as compared to controls (p<0.01)  NAFLD patients were 1.26 times more likely to be vitamin D deficient as compared to controls  Greater prevalence of vitamin D deficiency is seen in NAFLD patients (Figure 8) Eliades M et al. Aliment Pharmacol Ther.2013; 38: 246–254. CLINICAL EVIDENCE ON LOW LEVELS OF VITAMIN D IN DIABETIC PATIENTS WITH NAFLD

23.  Therefore, decreased serum 25(OH)D levels in NAFLD patients suggested that vitamin D deficiencymay play a role in the development of NAFLD  Furthermore, anti-inflammatory and immunomodulatory properties of vitamin D are known to impact progression and severity of NAFLD Eliades M et al. Aliment Pharmacol Ther.2013; 38: 246–254. CLINICAL EVIDENCE ON LOW LEVELS OF VITAMIN D IN DIABETIC PATIENTS WITH NAFLD

24.  Another study conducted by Barchetta I et al., evaluated the link between vitamin D deficiency and NAFLD in patients with various degrees of insulin- resistance and related metabolic disorders  The study results reported reduced serum 25(OH)D levels in patients with NAFLD as compared to patients without NAFLD (14.8 ± 9.2 vs. 20.5 ± 9.7 ng/mL; p<0.001)  This relationship between reduced 25(OH)D levels and NAFLD was reported to be independent of age, sex, triglycerides, high density lipoproteins (HDL) and glycaemia (p<0.005)  Also, Fatty Liver Index was reported to be inversely correlated with low 25(OH)D levels, irrespective of sex, age and HOMA-IR (p<0.007)  Therefore, this study demonstrated strong independent link between NAFLD and low serum levels of vitamin D Barchetta I et al. BMC Med. 2011;9:85. CLINICAL EVIDENCE ON LOW LEVELS OF VITAMIN D IN DIABETIC PATIENTS WITH NAFLD

25.  In a study conducted by Kucukazman M et al., conducted among 154 patients with NAFLD and 57 patients without NAFLD, significantly lower levels of vitamin D in NAFLD patients as compared to control group were reported (p<0.0001; Figure 9)  Furthermore, increase in NAFLD grade was associated with gradual decline in serum levels of vitamin D Kucukazman M et al. Clinics (Sao Paulo). 2014;69(8):542-546. CLINICAL EVIDENCE ON LOW LEVELS OF VITAMIN D IN DIABETIC PATIENTS WITH NAFLD

26.  The role of vitamin D as an immunoregulatory nutrient has been documented in several studies 1  Several studies have documented association of vitamin D deficiency with insulin resistance, metabolic syndrome and NAFLD 1  Significant correlation has been reported with vitamin D deficiency and severe NAFLD activity score and hepatic fibrosis 1  Strong association has been reported between NAFLD and vitamin D deficiency, prediabetes, and type 2 diabetes 2  Studies have suggested an effective and promising role of vitamin D supplementation in patients with diabetes mellitus 2 1.Eslamparast T et al. World J Hepatol. 2015;7(2):204-212. 2.Nwosu BU, et al. PLoS ONE. 9(6): e99646. VITAMIN D SUPPLEMENTATION IN DIABETIC PATIENTS FOR TREATING NAFLD

27.  A study by Nwosu BU et al., was conducted to evaluate the hypothesis for improvement in vitamin D status, reduced hepatic dysfunction, and improved glycaemic control with vitamin D supplementation in children and adolescents with diabetes mellitus  This study determined the effect of vitamin D supplementation on ALT, HbA 1c, and 25(OH)D in patients with vitamin D deficiency and either type 1 diabetes or type 2 diabetes  In this study, 88 type 1 diabetic patients and 43 type 2 diabetic patients were included who received vitamin D supplementation Nwosu BU, et al. PLoS ONE. 9(6): e99646. VITAMIN D SUPPLEMENTATION IN DIABETIC PATIENTS FOR TREATING NAFLD

28.  Low levels of vitamin D were reported in 37.5% of T1D patients and 72.1% of patients with T2D (p<0.001)  Higher values for BMI SDS (p=0.001), ALT (p=0.001), but lower 25(OH)D (p<0.001) were reported in T2D patients as compared to T1D patients  Vitamin D supplementation after 3 months reported: o Significant increase in 25(OH)D in both T2D (p=0.015), and T1D patients (p<0.001) o Significant reduction in BMI SDS (p=0.015) in T2D than T1D patients o Significant reduction in ALT (p=0.012) in T2D, than T1D patients o Clinically-significant decrease in HbA 1c in T2D from 8.5 ± 2.9% at baseline to 7.7 ± 2.5 at 3 months, as compared to T1D, 8.5 ± 1.2 to 8.53 ± 1.1% Nwosu BU, et al. PLoS ONE.9(6): e99646. VITAMIN D SUPPLEMENTATION IN DIABETIC PATIENTS FOR TREATING NAFLD

29. o ALT decreased from 18.0 ± 6.0 U/L to 15.7 ± 2.0 (p = 0.46) in patients with type 1 diabetes; and from 50.7 ± 50.9 to 35.0 ± 31.0 (p = 0.012) in those with type 2 diabetes (Figure 10)  This suggested effective management of hepatic dysfunction as seen in NAFLD patients with diabetes  Therefore, vitamin D supplementation is effective to decrease ALT associated with vitamin D deficiency Nwosu BU, et al. PLoS ONE. 9(6): e99646. VITAMIN D SUPPLEMENTATION IN DIABETIC PATIENTS FOR TREATING NAFLD

30.  Non-alcoholic fatty liver disease (NAFLD) has been regarded as the commonest cause of chronic liver disease  In general Indian population, epidemiological studies suggested the prevalence of NAFLD to be 9% to 32%  Higher incidence has been reported in Indian patients who are overweight/obese and diabetic/prediabetic  Prevalence of NAFLD was reported to be 56.5%, across India in diabetic patients  NAFLD is associated with several conditions, including: o Obesity (69  100%) o Diabetes mellitus (36  75%) o Hyperlipidemia (20  81%)  Diabetes mellitus is reported in about 75% patients with NASH SUMMARY

31.  NAFLD is most common liver disease seen in diabetic patients, and it has been reported that 20% to 50% patients with NAFLD have diabetes  DM and NAFLD demonstrate common pathogenic link i.e., insulin resistance, both hepatic as well as peripheral  Several studies have demonstrated that vitamin D deficiency status may be responsible for the development and progression of NAFLD  Vitamin D supplementation has shown promising role in patients with diabetes mellitus in managing hepatic dysfunction SUMMARY