Research and Clinics Partnership Stress and Diabetes Mellitus (DM) at Cell and Molecular Levels

Introduction

Stress is the body’s response to any risk or requirements and recognised as an unfavourable event and a pathological change in the living tissue. It may vary from physical to emotional instabilities, often culminating into a wide range of ailments ranging from the decline in functional efficiency of organ systems to their failure. All these events are the result of micro-changes in cells, the minimum functional unit of the human body. Several aspects of biology aremimicked at the cellular level, hence considered a reliable in vitro model system not only to understand the normal and stressed signalling but also for drug discovery. These could be systematically studied and analysed in the laboratory.

Biology laboratory

Cellular and molecular biology laboratory is equipped with robust tools, techniques, reagents to understand the mechanism(s) of biological phenomena, and minds to access the clinical databases and reciprocate in the form of scientific advancement, better medicines and diagnostic devices. Such a laboratory accommodates operations ranging from cell culture to animal studies and extending to clinical trials in collaboration with medical institutions.

Cell culture studies

Cell culture studies use a wide variety of assays such as morphological observations and cytotoxicity evaluation to investigate the underlying molecular mechanism(s) that have been established as extremely powerful and economic step in the prototype development for drug discovery, along walk from bench to bedside. The essence of this concept is the fact that the cells respond to altered environment and stress precisely in ways that collectively constitute the whole-body response. Some of the best examples are:

(i) Normal cells age in culture, stop dividing at pre-determined population doublings, and show markers that have obtained consensus within-vivo phenotypes,

(ii) Cells from premature ageing syndromes show shorter life-span in culture, and

(iii) Stressed cells show premature ageing and express molecular markers of old age including shorter telomeres and elevated expression of tumour suppressor and stress marker proteins.

Last three decades have generated sufficient data to establish cell culture as a reliable tool for drug discovery regimes, and routinely utilised for drug discovery, development and validation of research findings.

Diabetes mellitus

Diabetes mellitus (DM) is a kind of stressful situation, a chronic condition where one’s body is unable to utilise the energy reserves present efficiently. At present, over 425 million people around the world and 72 million people in India are suffering from DM. India, now designated as the potential diabetic capital of the world. In DM, the living cells are unable to uptake glucose for energy production due to insufficient availability of functional insulin hormone. This leads to a stressful state within the cells, leading to the abnormal cellular signalling of proteins and may result in dysfunctions such as heart and brainrelated diseases, blindness, liver or kidney failure.

DM is largely defined as a group of diseases distinguished by a consistent increase in blood sugar resulting from inadequate insulin secretion, action, or both [5]. Various mechanisms of action are attributed to it, such as destruction of insulinproducing cells in the pancreas leading to insulin deficiency or abnormal reception of normal insulin (or its antagonist glucagon). Clinically, normal blood glucose measures are 0.7-1.0 mg/ ml (fasting) and 1.0-1.4 mg/ml (post-prandial - taken 2 hours after the meal). Increased blood sugar in diabetic patients is often associated with symptoms including increased frequency of urination, excessive thirst, weight loss, hunger and blurry vision. Long-term complications of DM include eye-related disorders and blindness, kidney failure, nerve-related diseases resulting in ulcers, amputations and joint deformities. Also, heart- and brain-related abnormalities, and sexual problems are encountered commonly.

Classification

DM is broadly classifiedas type1, type 2 and others [5]. Type 1 disease characterised by destruction of insulin secreting β-cells leading to absolute deficiency of insulin. Type2 diabetes includes a host of conditions resulting from resistance to sense insulin or a secretory defect of receptors. Other types result from genetic defects, obesity, injury, rupture, cancer, drugreactions, infections, and various other pathologies. Gestational diabetes is a particular type of DM originates during the pregnancy and defined by any degree of glucose intolerance. Individuals who have not yet established DM, but are on the verge, are considered as ‘at risk’, or ‘pre-diabetic’ population. These are clinically diagnosed and labelled by impaired tolerance to glucose, i.e., blood glucose level ranging from1.0-1.26 mg/ml (fasting), 1.42.0 mg/ml (post-prandial), or impaired HbA1c enzyme standards (5.7-6.4%).

DM has been linked directly with the mitochondria, the powerhouse of cells, dysfunction in the host cells. Clinically, control of diabetes has been well related to the maintenance of a healthy lifestyle that includes regular exercise and healthy state of body weight with proper diet. Measures to prevent obesity and promotion of the concept of small frequent meals are generally the first advise from the clinicians to the patients. Modern medicines have played a tremendous role in controlling the disease.

Metformin

Metformin is the most widely prescribed and accepted orally administered medicine to manage diabetes. Its mechanism of action mainly revolves around the alteration of liver glucose output. It was shown to inhibit mitochondrial oxidation, thus enhancing insulin sensitivity and increasing glucose digestive enzymes. It also acts on the intestines to improve glucose utilisation and enhances GLP-1 (glucagon-like peptide-1, an antiinsulin hormone) secretion and gut fauna. Origin of metformin as an anti-diabetic molecule dates back to the 18th and 19th century, an excellent result of its laboratory-based extraction and exploration from the natural sources.

Since then, various studies have been conducted on the toxicity profile of the medicine and its role in other diseases. One of the many meta-analyses and reviews reported that metformin was safe in clinical trials and prevented the rate of conversion of pre-diabetes to diabetes by about 45%. Similarly, many other molecules have been introduced into the market and have occupied a wide position in the field of pharmaceuticals -glucosidase inhibitors, exenatide (GLP-1 analogues), and nateglinide (insulin secretagogues). Apart from these, various adjuvant therapies such as anti-obesity drugs, antihypertensives, gastro-surgical interventions, have also attained the limelight.

Stress proteins

Many proteins present in the cells and often listed on the blood profile reportare are implicated in the pathogenesis of the disease [6]. These include glutamine fructose-6-phosphate amidotransferase (GFAT), tyrosine phosphatase (PTPs), 11β-hydroxysteroid dehydrogenase, glucagon and insulin itself. Although recognisedin the stressed state of cells and tissues, the role of stress proteins is less studied and acknowledged in diabetes pathology.

Mortalin

One of the essential proteins suggested to be associated with the pathophysiology of DM, especially type I, is Mortalin. Mortalin protein plays a prominent role in control of cell proliferation. It has multiple functions, and is essential for vital processes including mitochondria genesis, oxidation and energy generation, and transporting proteins through the mitochondrial membrane. Mortalin is enriched in a large variety of cancers and has been shown to promote cancerous properties of cells from immortalisation to metastasis. Antimortalin antibody is readily detected in the blood samples of the patients diagnosed with liver cirrhosis and cancer. It has also been demonstrated that a high concentration (>60 ng/ml) of mortalin in blood is a significant risk factor for reduced overall survival and associated with poor disease prognosis.

The concurrent changes in mortalin and otherstress proteins including hsp70 (heat shock protein 70) often worsens the disease prognosis and hence have been proposed as drug targets for diseases including cancer and DM . In contrast to cancer, cells derived from old age pathologies including Alzheimer’s and Parkinson’s disease have been shown to display decreased expression of mortalin. Alzheimer’s disease is a long-standing neurodegenerative disease, characterized by deposition of amyloid -protein, gradual memory loss, behavioral problems, social detachment, and loss of motivation to live; Parkinson’s disease, on the other hand, is associated with deposition of Lewy bodies within the nerve cells, abnormal shaking, rigidity, difficulty to move or walk, loss of memory and fatal depression.In Alzheimer’s disease, cells were found to be deficient of mortalin that led to mitochondrial fragmentation and functional anomalies. Thus, interventions encircling mortalin expression and functions to maintain a healthy state of mitochondria were suggested to prevent/ delay the onset of the disorder.

About 38% of diabetic patients are indicated to have increased risk to develop Parkinson’s disease. Insulin resistance, i.e., type 2DM has been closely implicated to be central in the development of Parkinson’s disease. De Mena et al. screened brain samples from over 500 patients and found that mutations in the mortalin gene directly correlated with the risk of onset of Parkinson’s disease. Another study showed the preferential distribution of mortalin protein in different compartments of the brain cells. Within the Parkinson’s disease patient samples, mortalin was found to be expressed less in the cytoplasm of limbic cells, and in mitochondria of isocortex and limbic system cells. Overall, the level of mortalin protein was found to be decreased in the advanced Parkinson’s disease models.

Mortalin has also shown to be poorly expressed uniquely in the astrocytes in the substantia nigra pars compacta, an area in the basal ganglia of the brainstem responsible for movements in the Parkinson’s patients as compared to the normal. One of the essential proteins found in the Lewy bodies (characteristically expressed in Parkinson’s disease) is -synuclein.  It has been suggested to be a potential diagnostic marker in samples derived directly from Parkinson’s brain. Various studies, using animal models, have suggested that mortalin plays a vital role in preventing-synuclein-mediated brain toxicity in the pathogenesis of Parkinson’s disease. Recently, researchers demonstrated a negative correlation of mortalin with -synuclein and have suggested its role as a new and better diagnostic marker.

In DM, mortalin was found to be over-produced in rat pancreas (responsible for insulin sovereignty) kept under continuous stressful conditions. Regular pancreatic insulin-secreting cells are likely to mimic pre-diabetes with prolonged exposure to the stressors. When these cells were exposed to inflammation-facilitating compounds, it was noticed that there is an increase in secretion of mortalin protein. This increase was associated with lesser survival and expedited ageing, suggesting a potential role of mortalin in the pathogenesis of DM. Mortalin may also be related to the atypical expression of other critical diabetic hormones such as glucagon and C-peptide. Of note, whereas an increase in the overall concentration of mortalin was found to correlate with cancer, diabetes and other metabolic diseases, inactivation of its genes and down regulation has been associated with neurodegenerative diseases. Although a few studies have revealed that mortalin has a direct correlation with the onset and progression of DM, more detailed investigations are warranted.

All of the anti-diabetic therapies in practice available today are for diabetic control, but none to cure. Many studies in past around the world have analysed patient’s samples and have developed some of the first anti-diabetes medicines. Continuous feedback from the patients is crucial in the discovery and development of continuously improving, better efficacy and safe medications. Regrettably, at present, the data from the clinics of soon-to-be world diabetic capital (India) that reaches the laboratory for scientific studies is inadequate. There is immense potential for India to excel in the drug discovery programs and personalised therapy, especially for diabetes. Diagnosis and treatment are incomplete without research and development. Practices on regular submission and analysis of patient samples, networking and feedback with laboratory experts are deemed to be very useful and yield new seeds and leads. If you are suffering from diabetes mellitus, trust that you are not alone, your crosstalk with the biomedical researchers may help to find new ways to deal with it.