Liver fat in the metabolic syndrome and type 2 diabetes

Introduction: The epidemic of obesity has been accompanied by an increase in the prevalence of the metabolic syndrome, type 2 diabetes, and non-alcoholic fatty liver disease (NAFLD). However, not all obese subjects develop these metabolic abnormalities. Hepatic fat accumulation is related to hepatic insulin resistance, which in turn leads to hyperglycemia, hypertriglyceridemia, and a low HDL cholesterol con-centration. The present studies aimed to investigate 1) how intrahepatic as compared to intramyocellular fat is related to insulin resistance in these tissues and to the metabolic syndrome (Study I); 2) the amount of liver fat in subjects with and without the metabolic syndrome, and which clinically available markers best reflect liver fat content (Study II); 3) the effect of liver fat on insulin clearance (Study III); 4) whether type 2 diabetic patients have more liver fat than age-, gender-, and BMI-matched non-diabetic subjects (Study IV); 5) how type 2 diabetic patients using exceptionally high doses of insulin respond to addition of a PPARγ agonist (Study V). Subjects and methods: The study groups consisted of 45 (Study I), 271 (Study II), and 80 (Study III) non-diabetic subjects, and of 70 type 2 diabetic patients and 70 matched control subjects (Study IV). In Study V, a total of 14 poorly controlled type 2 diabetic patients treated with high doses of insulin were studied before and after rosiglitazone treatment (8 mg/day) for 8 months. In all studies, liver fat content was measured by proton magnetic resonance spectroscopy, and sub-cutaneous and intra-abdominal fat content by MRI. In addition, circulating markers of insulin resistance and serum liver enzyme concentrations were determined. Hepatic (i.v. insulin infusion rate 0.3 mU/kg∙min combined with [3-3H]glucose, Studies I, III, and V) and muscle (1.0 mU/kg min, Study I) insulin sensitivities were measured by the euglycemic hyperinsulinemic clamp technique. Results: Fat accumulation in the liver rather than in skeletal muscle was associated with features of insulin resistance, i.e. increased fasting serum (fS) triglycerides and decreased fS-HDL cholesterol, and with hyperinsulinemia and low adiponectin concentrations (Study I). Liver fat content was 4-fold higher in subjects with as compared to those without the metabolic syndrome, independent of age, gender, and BMI. FS-C-peptide was the best correlate of liver fat (Study II). Increased liver fat was associated with both impaired insulin clearance and hepatic insulin resistance independent of age, gender, and BMI (Study III). Type 2 diabetic patients had 80% more liver fat than age-, weight-, and gender-matched non-diabetic subjects. At any given liver fat content, S-ALT underestimated liver fat in the type 2 diabetic patients as compared to the non-diabetic subjects (Study IV). In Study V, hepatic insulin sensitivity increased and glycemic control improved significantly during rosiglitazone treatment. This was associated with lowering of liver fat (on the average by 46%) and insulin requirements (40%). Conclusions: Liver fat is increased both in the metabolic syndrome and type 2 diabetes independent of age, gender, and BMI. A fatty liver is associated with both hepatic insulin resistance and impaired insulin clearance. Rosi-glitazone may be particularly effective in type 2 diabetic patients who are poorly controlled despite using high insulin doses.

Studies of Immune Regulation in Type 1 Diabetes

Type 1 diabetes (T1D) is considered to be an autoimmune disease. In T1D insulin producing pancreatic β cells are destroyed. The disease process begins years before the clinical diagnosis of T1D. During the pathogenesis of T1D, pancreatic islets are infiltrated by cells of the immune system and T-lymphocytes are considered to be the main mediators of the β-cell destruction. In children with an active β-cell destruction process, autoantibodies against β-cell antigens appear in the blood. Individuals at increased risk of developing T1D can often be identified by detecting serum autoantibodies against β-cell antigens. Immunological aberrancies associated with T1D are related to defects in the polarization of T cells and in the function of regulatory mechanisms. T1D has been considered as an organ-specific autoimmune disease mediated by uncontrolled Th1-responses. In human T1D, the evidence for the role of over-expression of cytokines promoting cytotoxicity is controversial. For the past 15 years, regulatory T cells (Tregs) have been recognized as having a key role in the initiation and maintenance of tolerance, limiting harmful autoantigen-specific inflammation processes. It is possible that, if regulatory mechanisms fail to be initiated, the subtle inflammation targeting β cells lead to insulitis and eventually to overt T1D in some individuals. In the present thesis, we studied the induction of Tregs during the generation of T-cell responses in T1D. The results suggest that the generation of regulatory mechanisms and effector mechanisms upon T-cell activation is aberrant in children with T1D. In our studies, an in vitro cytotoxic environment inhibited the induction of genes associated with regulatory functions upon T-cell activation. We also found T1D patients to have an impaired cytotoxic response against coxsackievirus B4. Ineffective virus clearance may increase the apoptosis of β cells, and thus the risk of β-cell specific autoimmunity, due to the increased presentation of β-cell-derived peptides by APCs to T cells in pancreatic lymph nodes. Recently, a novel T helper cell subset called Th17 has been discovered. Animal models have associated Th17 cells and especially co-producers of IL-17 and IFN-γ with the pathogenesis of T1D. We aimed to characterize the role of Th17 immunity in human T1D. We demonstrated IL-17 activation to be a major alteration in T1D patients in comparison to healthy children. Moreover, alterations related to the FOXP3-mediated regulatory mechanisms were associated with the IL-17 up-regulation seen in T1D patients. These findings may have therapeutic implications for the treatment and prevention of T1D.

Glycaemic behaviour during breastfeeding in women with Type 1 diabetes

Aim To describe glycaemia in both breastfeeding women and artificially feeding women with Type 1 diabetes, and the changes in glycaemia induced by suckling. Methods A blinded continuous glucose monitor was applied for up to 6 days in eight breastfeeding and eight artificially feeding women with Type 1 diabetes 2–4 months postpartum. Women recorded glucose levels, insulin dosages, oral intake and breastfeeding episodes. A standardized breakfast was consumed on 2 days. A third group (clinic controls) were identified from a historical database. Results Carbohydrate intake tended to be higher in breastfeeding than artificially feeding women (P = 0.09) despite similar insulin requirements. Compared with breastfeeding women, the high blood glucose index and standard deviation of glucose were higher in artificially feeding women (P = 0.02 and 0.06, respectively) and in the clinical control group (P = 0.02 and 0.05, respectively). The low blood glucose index and hypoglycaemia were similar. After suckling, the low blood glucose index increased compared with before (P < 0.01) and during (P < 0.01) suckling. Hypoglycaemia (blood glucose < 4.0 mmol/l) occurred within 3 h of suckling in 14% of suckling episodes, and was associated with time from last oral intake (P = 0.04) and last rapid-acting insulin (P = 0.03). After a standardized breakfast, the area under the glucose curve was positive. In breastfeeding women the area under the glucose curve was positive if suckling was avoided for 1 h after eating and negative if suckling occurred within 30 min of eating. Conclusions Breastfeeding women with Type 1 diabetes had similar hypoglycaemia but lower glucose variability than artificially feeding women. Suckling reduced maternal glucose levels but did not cause hypoglycaemia in most episodes.

Assessment of Risk for Type 1 Diabetes in Children of Affected Families and in the General Population : Role of Immunological and Metabolic Markers

Background and aims. Type 1 diabetes (T1D), an autoimmune disease in which the insulin producing beta cells are gradually destroyed, is preceded by a prodromal phase characterized by appearance of diabetes-associated autoantibodies in circulation. Both the timing of the appearance of autoantibodies and their quality have been used in the prediction of T1D among first-degree relatives of diabetic patients (FDRs). So far, no general strategies for identifying individuals at increased disease risk in the general population have been established, although the majority of new cases originate in this population. The current work aimed at assessing the predictive role of diabetes-associated immunologic and metabolic risk factors in the general population, and comparing these factors with data obtained from studies on FDRs. Subjects and methods. Study subjects in the current work were subcohorts of participants of the Childhood Diabetes in Finland Study (DiMe; n=755), the Cardiovascular Risk in Young Finns Study (LASERI; n=3475), and the Finnish Type 1 Diabetes Prediction and Prevention Study (DIPP) Study subjects (n=7410). These children were observed for signs of beta-cell autoimmunity and progression to T1D, and the results obtained were compared between the FDRs and the general population cohorts. --- Results and conclusions. By combining HLA and autoantibody screening, T1D risks similar to those reported for autoantibody-positive FDRs are observed in the pediatric general population. Progression rate to T1D is high in genetically susceptible children with persistent multipositivity. Measurement of IAA affinity failed in stratifying the risk assessment in young IAA-positive children with HLA-conferred disease susceptibility, among whom affinity of IAA did not increase during the prediabetic period. Young age at seroconversion, increased weight-for-height, decreased early insulin response, and increased IAA and IA-2A levels predict T1D in young children with genetic disease susceptibility and signs of advanced beta-cell autoimmunity. Since the incidence of T1D continues to increase, efforts aimed at preventing T1D are important, and reliable disease prediction is needed both for intervention trials and for effective and safe preventive therapies in the future. Our observations confirmed that combined HLA-based screening and regular autoantibody measurements reveal similar disease risks in pediatric general population as those seen in prediabetic FDRs, and that risk assessment can be stratified further by studying glucose metabolism of prediabetic subjects. As these screening efforts are feasible in practice, the knowledge now obtained can be exploited while designing intervention trials aimed at secondary prevention of T1D.

Innate immune system and adiponectin in diabetic nephropathy in type 1 diabetes

Introduction: The pathogenesis of diabetic nephropathy remains a matter of debate, although strong evidence suggests that it results from the interaction between susceptibility genes and the diabetic milieu. The true pathogenetic mechanism remains unknown, but a common denominator of micro- and macrovascular complications may exist. Some have suggested that low-grade inflammation and activation of the innate immune system might play a synergistic role in the pathogenesis of diabetic nephropathy. Aims of the study: The present studies were undertaken to investigate whether low-grade inflammation, mannan-binding lectin (MBL) and α-defensin play a role, together with adiponectin, in patients with type 1 diabetes and diabetic nephropathy. Subjects and methods: This study is part of the ongoing Finnish Diabetic Nephropathy Study (FinnDiane). The first four cross-sectional substudies of this thesis comprised 194 patients with type 1 diabetes divided into three groups (normo-, micro-, and macroalbuminuria) according to their albumin excretion rate (AER). The fifth substudy aimed to determine whether baseline serum adiponectin plays a role in the development and progression of diabetic nephropathy. This follow-up study included 1330 patients with type 1 diabetes and a mean follow-up period of five years. The patients were divided into three groups depending on their AER at baseline. As a measure of low-grade inflammation, highly sensitive CRP (hsCRP) and α-defensin were measured with radio-immunoassay, and interleukin-6 (IL-6) with high- sensitivity enzyme immuno-assay. Mannan-binding lectin and adiponectin were determined with time-resolved immunofluorometric assays. The progression of albuminuria from one stage to the other served as a measure of the progression of diabetic nephropathy. Results: Low-grade inflammatory markers, MBL, adiponectin, and α-defensin were all associated with diabetic nephropathy, whereas MBL, adiponectin, and α-defensin per se were unassociated with low-grade inflammatory markers. AER was the only clinical variable independently associated with hsCRP. AER, HDL-cholesterol and the duration of diabetes were independently associated with IL-6. HbA1c was the only variable independently associated with MBL. The estimated glomerular filtration rate (eGFR), AER, and waist-to-hip ratio were independently associated with adiponectin. Systolic blood pressure, HDL-cholesterol, total cholesterol, age, and eGFR were all independently associated with α-defensin. In patients with macroalbuminuria, progression to end-stage renal disease (ESRD) was associated with higher baseline adiponectin concentrations. Discussion and conclusions: Low-grade inflammation, MBL, adiponectin, and defensin were all associated with diabetic nephropathy in these cross-sectional studies. In contrast however, MBL, adiponectin, and defensin were not associated with low-grade inflammatory markers per se. Nor was defensin associated with MBL, which may suggest that these different players function in a coordinated fashion during the deleterious process of diabetic nephropathy. The question of what causes low-grade inflammation in patients with type 1 diabetes and diabetic nephropathy, however, remains unanswered. We could observe in our study that glycemic control, an atherosclerotic lipid profile, and waist-to-hip ratio (WHR) were associated with low-grade inflammation in the univariate analysis, although in the multivariate analysis, only AER, HDL-cholesterol, and the duration of diabetes, as a measure of glycemic load, proved to be independently associated with inflammation. Notably, all these factors are modifiable with changes in lifestyle and/or with a targeted medication. In the follow-up study, elevated serum adiponectin levels at baseline predicted the progression from macroalbuminuria to ESRD independently of renal function at baseline. This observation does not preclude adiponectin as a favorable factor during the process of diabetic nephropathy, since the rise in serum adiponectin concentrations may remain a mechanism by which the body compensates for the demands created by the diabetic milieu.

The impact of the metabolic syndrome and parental risk factors in patients with type 1 diabetes

Background: One-third of patients with type 1 diabetes develop diabetic complications, such as diabetic nephropathy. The diabetic complications are related to a high mortality from cardiovascular disease, impose a great burden on the health care system, and reduce the health-related quality of life of patients. Aims: This thesis assessed, whether parental risk factors identify subjects at a greater risk of developing diabetic complications. Another aim was to evaluate the impact of a parental history of type 2 diabetes on patients with type 1 diabetes. A third aim was to assess the role of the metabolic syndrome in patients with type 1 diabetes, both its presence and its predictive value with respect to complications. Subjects and methods: This study is part of the ongoing nationwide Finnish Diabetic Nephropathy (FinnDiane) Study. The study was initiated in 1997, and, thus far, 4,800 adult patients with type 1 diabetes have been recruited. Since 2004, follow-up data have also been collected in parallel to the recruitment of new patients. Studies I to III have a cross-sectional design, whereas Study IV has a prospective design. Information on parents was obtained from the patients with type 1 diabetes by a questionnaire. Results: Clustering of parental hypertension, cardiovascular disease, and diabetes (type 1 and type 2) was associated with diabetic nephropathy in patients with type 1 diabetes, as was paternal mortality. A parental history of type 2 diabetes was associated with a later onset of type 1 diabetes, a higher prevalence of the metabolic syndrome, and a metabolic profile related to insulin resistance, despite no difference in the distribution of human leukocyte antigen genotypes or the presence of diabetic complications. A maternal history of type 2 diabetes, seemed to contribute to a worse metabolic profile in the patients with type 1 diabetes than a paternal history. The metabolic syndrome was a frequent finding in patients with type 1 diabetes, observed in 38% of males and 40% of females. The prevalence increased with worsening of the glycemic control and more severe renal disease. The metabolic syndrome was associated with a 3.75-fold odds ratio for diabetic nephropathy, and all of the components of the syndrome were independently associated with diabetic nephropathy. The metabolic syndrome, independent of diabetic nephropathy, increased the risk of cardiovascular events and cardiovascular and diabetes-related mortality over a 5.5-year follow-up. With respect to progression of diabetic nephropathy, the role of the metabolic syndrome was less clear, playing a strong role only in the progression from macroalbuminuria to end-stage renal disease. Conclusions: Familial factors and the metabolic syndrome play an important role in patients with type 1 diabetes. Assessment of these factors is an easily applicable tool in clinical practice to identify patients at a greater risk of developing diabetic complications.

Long-term effects of fenofibrate on lipoproteins and surrogate markers of macrovascular and microvascular disease in people with type 2 diabetes

Background and aims. Diabetic dyslipidemia is a highly atherogenic triad of increased triglycerides, decreased HDL cholesterol, and small dense LDL. Fibrates have a beneficial effect on diabetic dyslipidemia, and they have reduced cardiovascular events in randomized trials. Fenofibrate has reduced albuminuria and markers of low-grade inflammation and endothelial dysfunction. The present studies were undertaken to characterize the alterations of VLDL and LDL subclasses and to investigate the binding of LDL to arterial wall in type 2 diabetes. Further purpose was to elucidate the effects of fenofibrate on several lipoprotein subclasses, augmentation index (AIx), carotid intima-media thickness (IMT), and renal function. Subjects. 239 type 2 diabetic subjects were recruited among participants of the FIELD (Fenofibrate Intervention and Event Lowering in Diabetes) study at the Helsinki centre. The patients were randomized to fenofibrate (200mg/d) or placebo for 5 years. Additionally, a healthy control group (N = 93) was recruited. Results. VLDL1 triglycerides increased in similar proportion to total triglycerides in type 2 diabetic patients and control subjects. Despite the increase in total apoCIII levels, VLDL apoCIII was decreased in diabetic patients. Enrichment of LDL with apoCIII induced a small increase in binding of LDL to arterial wall proteoglycan. Intrinsic characteristics of diabetic LDL, rather than levels of apoCIII, were responsible for increased proteoglycan binding of diabetic LDL with high apoCIII. Fenofibrate reduced triglycerides, increased LDL size, and shifted HDL subclasses towards smaller particles with no change in levels of HDL cholesterol. High levels of homocysteine were associated with lower increase of HDL cholesterol and apoA-I during fenofibrate treatment. Long-term fenofibrate treatment did not improve IMT, AIx, inflammation, or endothelial function. Fenofibrate decreased creatinine clearance and estimated glomerular filtration rate. No effect on albuminuria was seen with fenofibrate. Instead, Cystatin C was increased during fenofibrate treatment. Conclusions. 1) Elevation of VLDL 1 triglycerides was the major determinant of plasma triglyceride concentration in control subjects and type 2 diabetic patients. 2) LDL with high apoCIII showed multiple atherogenic properties, that were only partially mediated by apoCIII per se in type 2 diabetes 3) Fenofibrate demonstrated no effect on surrogate markers of atherosclerosis. 4) Fenofibrate had no effect on albuminuria and the observed decrease in markers of renal function could complicate the clinical surveillance of the patients. 5) Fenofibrate can be used to treat severe hypertriglyceridemia or in combination therapy with statins, but not to increase HDL levels.

Effects of Metabolic Risk Factors and Type 1 Diabetes on Brain Glucose and Brain Metabolites

The metabolic syndrome and type 1 diabetes are associated with brain alterations such as cognitive decline brain infarctions, atrophy, and white matter lesions. Despite the importance of these alterations, their pathomechanism is still poorly understood. This study was conducted to investigate brain glucose and metabolites in healthy individuals with an increased cardiovascular risk and in patients with type 1 diabetes in order to discover more information on the nature of the known brain alterations. We studied 43 20- to 45-year-old men. Study I compared two groups of non-diabetic men, one with an accumulation of cardiovascular risk factors and another without. Studies II to IV compared men with type 1 diabetes (duration of diabetes 6.7 ± 5.2 years, no microvascular complications) with non-diabetic men. Brain glucose, N-acetylaspartate (NAA), total creatine (tCr), choline, and myo-inositol (mI) were quantified with proton magnetic resonance spectroscopy in three cerebral regions: frontal cortex, frontal white matter, thalamus, and in cerebellar white matter. Data collection was performed for all participants during fasting glycemia and in a subgroup (Studies III and IV), also during a hyperglycemic clamp that increased plasma glucose concentration by 12 mmol/l. In non-diabetic men, the brain glucose concentration correlated linearly with plasma glucose concentration. The cardiovascular risk group (Study I) had a 13% higher plasma glucose concentration than the control group, but no difference in thalamic glucose content. The risk group thus had lower thalamic glucose content than expected. They also had 17% increased tCr (marker of oxidative metabolism). In the control group, tCr correlated with thalamic glucose content, but in the risk group, tCr correlated instead with fasting plasma glucose and 2-h plasma glucose concentration in the oral glucose tolerance test. Risk factors of the metabolic syndrome, most importantly insulin resistance, may thus influence brain metabolism. During fasting glycemia (Study II), regional variation in the cerebral glucose levels appeared in the non-diabetic subjects but not in those with diabetes. In diabetic patients, excess glucose had accumulated predominantly in the white matter where the metabolite alterations were also the most pronounced. Compared to the controls values, the white matter NAA (marker of neuronal metabolism) was 6% lower and mI (glia cell marker) 20% higher. Hyperglycemia is therefore a potent risk factor for diabetic brain disease and the metabolic brain alterations may appear even before any peripheral microvascular complications are detectable. During acute hyperglycemia (Study III), the increase in cerebral glucose content in the patients with type 1 diabetes was, dependent on brain region, between 1.1 and 2.0 mmol/l. An every-day hyperglycemic episode in a diabetic patient may therefore as much as double brain glucose concentration. While chronic hyperglycemia had led to accumulation of glucose in the white matter, acute hyperglycemia burdened predominantly the gray matter. Acute hyperglycemia also revealed that chronic fluctuation in blood glucose may be associated with alterations in glucose uptake or in metabolism in the thalamus. The cerebellar white matter appeared very differently from the cerebral (Study IV). In the non-diabetic men it contained twice as much glucose as the cerebrum. Diabetes had altered neither its glucose content nor the brain metabolites. The cerebellum seems therefore more resistant to the effects of hyperglycemia than is the cerebrum.